Life Science Potluck

A potluck of discoveries, techniques and theories from a variety of life sciences.

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Undergrad Research Tips: Lab Work

Transitioning from academic research to industrial research has been an exciting experience. Of course, I suffered from imposter syndrome when I started my first job—but at some point, I realised: Oh my god, I actually have the skills to do my job.

I went from studying anemone toxins to characterising antibodies. My undergrad research experience helped me transition into my new job. A lot of the soft skills, core knowledge, and technical skills came in handy.   In my first post on undergrad research, I mentioned there were three main parts: literature review, lab work, and writing.  I've covered the literature review section, so I will pick up from last time and talk about lab work.

You can read my previous undergrad research tips here:

https://lifesciencepotluck.tumblr.com/post/174020398950/undergrad-research-tips-the-big-three

https://lifesciencepotluck.tumblr.com/post/178247479510/undergrad-research-tips-literature-review

Have an actual plan: I can't emphasise the importance of planning your undergrad research project. It's frustrating to meander around for a year and then rush writing a report or thesis at the last minute. Sit down with your mentor to come up with a flow chart of experiments and a timeline for your project. Set deadlines for data collection, data processing, and writing the first draft of your thesis. You will always need more time to write than you think. Sometimes, your strategy or experiment won't work. Discuss a backup plan with your PI for your project.

Maintain a good lab notebook: Keeping a good lab note is the first step to doing proper research. Every time you run an experiment or learn a new technique, write it in your lab notebook. You should include the date, the aim, the method and the results in your entries. Make clear notes on how you analyse your data, general observations during the experiment, and any deviations from the procedure. For example, maybe you saw a few particles in your protein sample, which could impact your results—write that down. If you're not a fan of pen and paper, OneNote or Evernote is a good alternative. I recommend you read this post on lab notebooks: https://www.sciencemag.org/careers/2019/09/how-keep-lab-notebook

Back up and document everything: Computers crash; thumb drives vanish. If your data is that precious, then you should back it up. Maybe you're using a mass spec, and you spent hours analysing your data. Don't think to yourself, 'I'll take the final processed spectra, it's not like I'll need the raw mass spec data.' You will need the raw data if you misinterpreted the data or you processed the raw data the wrong way. Keep copies of the raw and processed data just in case.

Double-check your calculations: Sometimes, something as small as calculation errors and preparing the wrong concentration of reagents can screw up your experiments. Always double check your calculations. Did you use the right volume or formula? Did you convert the units properly? If you can't calculate things on the spot and start your experiment right away, it's okay. If you're preparing tonnes of samples, use an excel sheet. Instead of, you know impatiently jamming numbers in the calculator, forgetting something or accidentally erasing something and then recalculating things for the fifth time.

Read and annotate the SOPs: A lot of labs will have their own SOPs, which give you step-by-step instructions for conducting specific experiments. You can highlight important conditions like incubation temperature and times. You can also and make notes on why the SOP has specific reagents and steps. You can include a flow chart to summarise all the steps in the experiment. Most importantly, you can use pretty colours!

Prepare for each experiment: When you use a machine or do an experiment for the first few times, you will have to refer to your notes, and that's okay. It's safer to check your notes than accidentally wrecking expensive equipment. I like to make a checklist of things of the critical steps, and I tick them off as I go along, so I don't forget anything. It's a good idea to make a 'do not' list, where you list possible things, which would damage the equipment or the ruin experiment. Keep referring to list, until it's lodged into your subconscious and you have vivid nightmares about forgetting to balance the centrifuge, before pressing start.

Do things the proper way: There is a difference between doing something and doing something properly. There is a proper way to weigh things; there is a proper way to pipette samples. Pipetting errors can ruin experiments, especially when getting concentration of your sample right is critical. The gist of pipetting is you press the plunger thingy, suck up a bunch of junk, press the plunger again and dump your junk in another tube. Of course, there's more to pipetting, from your speed to pipetting angle. Students should learn good pipetting practices early on, so proper pipetting becomes a habit. There's are tonnes of websites with GPP tips. Here's one of my favourites: https://www.biosistemika.com/blog/tips-to-improve-pipetting-technique/

Read the manual and learn about the technique: It takes months, sometimes years of practice to master a technique or instrument. Hands-on training is vital for learning any lab technique, but it's crucial to understand the theory as well. Start with the manual, understand the anatomy of the machine, and screenshot the troubleshooting guide. Once you know the basics, you can read papers, which dive into the strengths, limitations and blind spots for each technique. There's someone out there who has dedicated their entire life to developing and optimising one method. I may not have the same of expertise with CD spectrometer as a physicist. However, having a deeper understanding of the physics behind the technique, will help me with interpreting the data and making most of the technique.

Play with the software: A lot of instruments like mass spec or HPLC, have snazzy software, which allows you to run experiments and analyse data. One of the best ways of learning how to use these kinds of software is by playing around with the tools and exploring all of the options and icons. If you get stuck or you're curious about a specific feature, you can use the help option. Most technical software will have help guides within the software, with an index containing a variety of topics. Ask your mentors if there are spare data files, which you can use for practising data analysis. The first few times you analyse data, it's easy to have a lot of doubt and uncertainty about your data interpretation. As you analyse more data, you become more confident and better at spotting very subtle details. Sometimes, the manufacturer will have a series of videos on how to use their software. When I was learning how to analyse data for DSC, the TA instruments YouTube channel was a lifesaver.

Take care of yourself: I have seen a lot of students joking about how they stay overnight in the lab, skip lunches, and spend every spare waking moment running experiments on a daily basis —none of this is healthy. It's so easy to isolate yourself from your support system and neglect your wellbeing as you become engrossed in your project. The most important thing you have as a researcher and a student is your mind and your body—take care of yourself.

Initially, I was going to write one post about report writing, but I have really gotten into scientific and technical writing. So, you can expect more posts about scientific writing.

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The most important relationship ever

Previously on Anemone Toxins 201:

Last time I covered how we discover novel anemone toxins, determine the structure and figure out the function. Now, everything comes together because we're looking at the most important relationship ever: the structure-function relationship.

We know the structure of our anemone toxin; we know what the toxin binds to—but how does an anemone toxin bind to its target? What kind of exciting interactions are happening between a toxin and its target? Protein structure and function are like soulmates: protein function relies on the structure, and protein structure adapts to a protein's functional needs. It's essential to understand the structure-function relationship of animal toxins, before developing therapeutic proteins. Once we understand which amino acids are critical for binding to the receptor, it's easier to tweak the toxin to improve its stability, specificity, and affinity.

I won't cover every single approach for exploring the structure-function relationship of anemone toxins, but I want to highlight some of the most common strategies. 

Chemical Modifications

It's funny how Biologists learn about living things by screwing things up and seeing what happens. Let's change a couple of critical amino acids in this protein. Let's knock out a possibly important gene. Let's expose to rat to radiation! What could possibly go wrong? The 'screw it up and see' approach is the basis for chemically modifying amino acids or mutating specific amino acids.

Amino acids aren't forever: they are prone to degrading and forming weird side products. For chemical modifications, we don't change the protein sequence. Instead, we use a reagent or stress condition to modify the side chains. For mutagenesis, we introduce substitutions: usually, the target amino acid is replaced with alanine, and then we express the protein. Turk et al. (1989), chemically modified arginine with 2,3 butane-dione and they modified tyrosine with tetranitromethane. They found tyrosine was important for Equinatoxin II's haemolytic activity. The toxin with the modified tyrosine residues had lower haemolytic activity compared to the native form. However, using chemical modifications gives very general information about the type of amino acid involved in binding. It's hard to determine which specific modified amino acid, directly binds to the receptor, does nothing, or maintains the structure.

Figure 1: 3-D structure of Equinatoxin II. Tyrosine side chains are in red (PDB ID: 1IAZ)

Mutagenesis

A lot of recent papers use site-directed mutagenesis to target one amino acid at a time: it's precise and also laborious. Moran et al. used mutagenesis to figure out which amino acids in the sodium channel toxin, Av2 were involved in binding to  the Nav1.5 channel. They found that six residues were vital for binding and toxicity: valine-2, leucine-5, aspartic acid-9, asparagine-16, leucine-18, and isoleucine-41. Mutagenesis is excellent for pinpointing specific amino acids in a toxin. However, it's harder to figure out what kinds of biophysical interactions are happening between the toxin and receptor. Are there hydrogen bonds? Are there electrostatic interactions? Are there hydrophobic interactions?

X-ray crystallography

Sometimes, we can crystallise the toxin-receptor complex. The 3-D for the complex gives information about toxin-receptor interactions at an atomic level. Celie et al. managed to get the 3-D structure of a-conotoxin from cone snails (PnIA) bound to the nicotinic acetylcholine receptor (AChBP). They found that when PnIA binds to AChBP, PnIA shifts a specific loop (the C-loop).But, there's no significant conformational change in AChBP. The crystallised complex also showed that hydrophobic interactions were the main driver for PnIA-AChBP binding.

Sometimes, crystallising the toxin-receptor complex is not feasible. If you have the crystal structures of the toxin and receptors separately, you can mash the two puzzle pieces together by using molecular docking. The gist of molecular docking involves figuring how the two partners in a complex are orientated together. Tools like PatchDock, use the complementary shapes of two partners to predict the docked complex. Lanigan et al. (2002) used a combination of mutagenesis and docking to figure out how an analogue for ShK (ShK-Dap22) binds to potassium channel (Kv1.3). They found that ShK and ShK-Dap22 bind to Kv1.3 channel in different ways. ShK has a conserved, positively lysine residue, which blocks the negatively charged pore in the potassium channel. However, ShK-Dap22 doesn't bind to the same site as ShK. In fact, ShK-Dap22 binds closer to the mouth of the Kv1.3 channel.

Figure 2: 3-D structure of a-conotoxin-AChBP. The bound PnIA are color coded as brick red (PDB ID: 2BR7)

NMR

The crystal structure of a protein is like a photograph which captures one conformation—but proteins are dynamic and their conformations are in flux. NMR is suitable for monitoring protein dynamics, since it can pick up on changes in the environment of amino acids, e.g. polarity, hydrogen bonding, orientation, etc. Sometimes, we want to see if there are conformational changes in a receptor after the toxin binds to the receptor. Are there any changes in NMR absorption for specific amino acids within the receptor, after binding? Lange et al. used solid-state NMR to explore how kalotoxin (KTX) from Fattail scorpions blocks Kv1.3 potassium channels. They found that both KTX and Kv1.3 went through conformational changes after binding. KTX binding impacted amino acids in the pore and the selectivity filter of the Kv1.3 channel.

Figure 3: General structure for potassium channels (Source: https://commons.wikimedia.org/wiki/File:Potassium_channel.jpg) 

All of the approaches I've covered are not only useful for exploring the structure-function relation of anemones toxins, but proteins in general. I have gushed about the therapeutic potential of anemone toxins in my posts. So, next time, I will cover a real-life example of how drugs are developed from an anemone toxins.

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Figuring out function

Previously on Anemone Toxins 201:

We covered how sequencing and bioinformatic tools can identify novel toxins. We covered how to get the structure of anemone toxins. Now, I will discuss how assays can probe and prove the function of an anemone toxin. 

I will break the assays down based on different types of anemone toxins. I covered the basics of different kinds of anemone toxins in Anemone Toxins 101.

Toxicity and antimicrobial assay

Before we try out any assay, we have to purify the venom and isolate the fraction with our toxin of interest. Well, how do you know your fraction or venom extract is toxic? You inject the venomous concoction into a poor crab or shrimp. The toxicity assay has to be developed based on the anemone's prey. Some anemones prey on crabs; some anemones can take out mammals with their toxins. Typically, we inject the crude venom or the fraction into the prey and observe the response: death, paralysis, foaming or nothing at all.

A lot of anemone toxins also have antimicrobial activity, especially the pore-forming toxins. For the antimicrobial assay, the crude venom is prepared at different concentrations, the venom is mixed into the cell culture, and we see how it impacts microbial growth.

Pore-forming toxins

For pore-forming toxins like actinoporins and cytolysins,  haemolytic assay are the way to go. Can your novel toxin lyse blood cells like it's nobody's business? Just like the antimicrobial assay, we have to prepare different dilutions of the toxin. The hemolysis assay is super convenient because we can plate the red blood cells, add the toxin, and measure the absorbance. If the toxins lyse the blood cells, the haemoglobin from blood cells gets released. The release of haemoglobin is what gives us a change in absorbance. The assay also tells us about dose-dependence. Do you need a lot of the toxin to cause a bit hemolysis? Or is a very diluted sample enough to cause a lot of lysis? In short, haemolytic assays can identify pore-forming viruses, while measuring how effectively the toxin is at slices and dices cells.

Figure 1: Let our powers combine! Oligomeric structure for Frac. (Source: Rojko et al, 2016)

Phospholipase A2

Since both phospholipases and cytolysins wreck cell membranes, it makes sense to do a haemolytic assay for phospholipases as well. Wait—if we can use the same type of assay for cytolysins and phospholipases A2, how can someone tell the difference between these two types of toxins? The key here is the phospholipids are in the membrane and that's the primary target for phospholipases A2. We can use fluorescent probes to label different types of phospholipases within the membrane. Each membrane can be labelled for one type of phospholipid. If phospholipases A2 (PLA2) hydrolyses the membrane, then the fluorescently labelled phospholipids are set free from the membrane, and we can measure the change in fluorescence. This assay also helps pin down which type of phospholipid (e.g. phosphatidic acid or phosphatidylcholine) the PLA2 binds to specifically.

Figure 2: Fluorescein labelled phospholipid. (Source: Thermofisher, Molecular Probes for lipids and membranes Handbook) 

Ion channel toxins

Out of all the different types of anemone toxins, ion channel toxins are most challenging to characterise. Not only do we have different types of ion channels like sodium, calcium, chloride and potassium channels: we also have hundreds of families and subfamilies for each kind of ion channel. Each subtype can be found in different cells and have different functions. For example, KV1 channels are found practically everywhere in the body, with the Kv1.3 channels playing a vital role in the nervous and immune system. However, the Kv11.1 channel is only found in cardiac cells.

How do we figure out which channel our toxin binds to? How do you figure out if the toxin is a pore blocker or channel modifier?

The best way of figuring out function for ion channel toxins is with electrophysiological assay like patch clamps? The gist of patch clamps is that there is a single cell with one specific channel (e.g. Kv1.1) and you can measure changes in the current of the ion channel. Once you pipette the ion toxin into the cell, there will be a change in the current, because the toxins blocks or hinders the flow of ions. The channel modifiers will change the gating kinetics and alter the threshold. For example, the anemone toxins could lock the ion channel in the open state or closed state, which would change the kinetics. A pore-forming toxin will lower the current without changing the threshold or kinetics.

Figure 3: Patch clamp data for channel modifying toxin: AaHII (Source: Kalia et al, 2016)

Figure 4: Patch clamp data for bore blocking toxin: TTX (Source: Kalia et al, 2016)

It's important to have high throughput assays, where you can screen multiple channels at the same time. Yes, bioinformatics can give clues about function, but sequence homology can be misleading. A toxin can dress like a sodium channel toxin, but act as potassium channel toxin in practice. APETx1 and APETx2, are very similar to each other in terms of the sequence. For example, APETx1 blocks a potassium channel (i.e. Kv11.1); APETx2 blocks acid-sensing ion channels (i.e. ASIC3). They don't even block the same type of ion channel, but they have similar sequences. All because of a few minor differences in key functional amino acids, these toxins have entirely different functions.

In the next post,  I'll look at the most important, relationship ever—the structure-function relationship.

Sources:

Anderluh, G. & Mac, P. Cytolytic peptide and protein toxins from sea anemones ( Anthozoa : Actiniaria ). Toxicon 40, (2002).

Romero, L. et al. Enzymatic and structural characterization of a basic phospholipase A 2 from the sea anemone Condylactis gigantea. Biochimie 92, 1063–1071 (2010).

Kalia, J. et al. From foe to friend: using animal toxins to investigate ion channel function. J Mol Biol 427, 158–175 (2016).

Ghosh, S., Kumar, T. T. A. & Balasubramanian, T. Characterization and anti microbial properties from the sea anemones [ Heteractics magnifica and Stichodactyla mertensii ] toxins. 3, 109–117 (2011).

Messerli, S. M. & Greenberg, R. M. Cnidarian toxins acting on voltage-gated ion channels. Mar. Drugs 4, 70–81 (2006).

Honma, T. & Shiomi, K. Review Article Peptide Toxins in Sea Anemones : Structural and Functional Aspects. Mar. Biotechnol. 8, 1–10 (2006).

Jouiaei, M. et al. Ancient venom systems: A review on cnidaria toxins. Toxins (Basel). 7, 2251–2271 (2015).

Potrich, C. et al. Cytotoxic activity of a tumor protease-activated pore-forming toxin. Bioconjug. Chem. 16, 369–376 (2005).

Ros, U. et al. Differences in activity of actinoporins are related with the hydrophobicity of their N-terminus. Biochimie 116, 70–78 (2015).

Segev, A., Garcia-Oscos, F. & Kourrich, S. Whole-cell Patch-clamp Recordings in Brain Slices. J. Vis. Exp. 1–10 (2016). doi:10.3791/54024 https://www.thermofisher.com/sg/en/home/references/molecular-probes-the-handbook/probes-for-lipids-and-membranes.html

Rojko, N., Dalla Serra, M., MačEk, P. & Anderluh, G. Pore formation by actinoporins, cytolysins from sea anemones. Biochim. Biophys. Acta - Biomembr. 1858, 446–456 (2016).

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Figuring out structure

Proteins are like molecular machines. Unlike actual machines, proteins can fold into intricate 3-D structures on their own. Wouldn't it be convenient if your bookshelf from IKEA could do that? If we want to understand how proteins work, we need a blueprint. Some parts of the protein are essential for stability; other parts are crucial for getting things done through binding or catalysis. We need to understand how different parts of the protein fit together; this is where Structural Biology comes in. Getting the 3-D structure is one of the main steps in characterising new anemone toxins. I will cover some of the techniques we use to investigate the toxin structure.

The hierarchy of protein structure goes like this: primary structure-->secondary structure-->tertiary structure-->quaternary structure. For anemone toxins, we don't have to worry about the quaternary structure. Usually, anemone toxins have one polypeptide, not multiple polypeptides.

Figure 1. Structures of anemone toxins (Source: Norton, R. S, 2009)

Primary structure:

It's the amino acid sequence or 'code' for the protein. You can get the amino acid sequence from DNA, mRNA or protein sequencing. Protein sequencing just gives you the sequence for your protein. However, if we use DNA or mRNA sequencing, then we have to figure out the 'mature' peptide sequence. DNA and mRNA code for the 'first draft' of the peptide. After translating mRNA to protein, the signal peptide and pro-peptide are attached to the 'mature peptide.' Those two parts of the protein get cut out before our mature peptide can go out in the world and decimate prey.

Secondary structure:

Our flat polypeptide chain will fold into different secondary structures like alpha-helices, beta-sheets, beta turns, etc. Circular dichroism and Fourier transform infrared radiation (FT-IR) can tell us about the secondary structure of the toxin. CD and FT-IR are commonly used to predict the amount of each secondary structure in proteins. For CD, we measure how much of circularly polarised light in the far-UV region is absorbed by the sample. We use curve fitting programs to determine which combination of secondary structures fits the spectra the best (e.g. 50% beta-sheet, 10% alpha-helix, 20 % random coil and 20 % beta-turn). FT-IR works similarly, but we use infrared radiation instead of polarised light. FT-IR is a better option for investigating the interactions between pore-forming toxins and lipid membranes. The lipid vesicle or 'sacs' scatter polarised light, which it makes challenging to use CD for the same purpose.

One of the best things about spectroscopic techniques is that they are not as time-consuming as, X-ray crystallography. You can have multiple CD, and FT-IR runs in 1-2 hours and get the processed data before lunch. However, CD and FT-IR are low-resolution techniques. They don't tell you the exact position and orientation of tryptophan 32. We need atomic resolution structures to get an in-depth understanding of how anemone toxins work.

Tertiary structure:

The full 3-D structure of proteins will have secondary structure plus the disulphide linkages, salt bridges, hydrophobic interactions. There are two main ways of getting atomic resolution structures: X-ray crystallography and NMR. X-ray crystallography can give high-resolution 3-D structures—if you manage to please the crystal gods. If you don't have the ideal crystallisation conditions—no crystal for you. I know PhD students who spent 12-18 months on crystallising their protein. However, once you get past the crystallisation bottleneck, the diffraction experiments and data processing doesn't take as much time. In theory, you can get better resolution with X-ray crystallography than NMR. Let's assume you don't have time for crystallisation and you go with NMR. NMR is excellent for finding the structure of smaller proteins, but for larger proteins, you have to use isotopic labelling. Some proteins that are too big for NMR. In X-ray crystallography, the protein is a 'frozen' state. However, NMR also lets you study proteins in solution to understand the dynamics of the protein. You can observe how the protein changes when it binds to something.

We can also predict the 3-D structure of the toxin-based on the sequence alone.

How?

Computer magic !

With the protein data bank and structure prediction tools like SWISS-MODEL, you can predict the 3-D structure of a new toxin. If there is an anemone toxin in the database with an experimentally determined 3-D structure and it has high sequence similarity with your new toxin, you can use homolog modelling. If you can't use homology modelling, you can use de novo structure prediction like I-TASSER. In de novo modelling, the program predicts the structure for smaller sections of the protein and uses those bits to build the full predicted model. Model prediction isn't 100% accurate, and we still have to determine the 3-D structure experimentally. However, predicted 3-D structures act as a guide.

Every technique has its strengths and weaknesses. A good researcher tries to use the most suitable method for their project to its full potential. You have to realise the power that's inside.

So, what can you do once you have the 3-D structure of a toxin?

I am going to use APETx1 from the clonal anemone as an example. APETx1 is a potassium channel toxin, which targets the Ether-a-gogo channels in heart cells. Since APETx1 is a small potassium channel toxin, Chagot and company could use NMR. They found that APETx1 belong to the Defensin family. A variety of anti-bacterial proteins and ion channel toxins belong to this family. Proteins with Defensin-4 domain, have double or triple-stranded antiparallel b-sheets linked to a short a-helix by two disulphide bridges. Since NMR can reveal disulphide linkages, Chagot found APETx1 has an inhibitor cysteine knot (ICK). The inhibitor cysteine is a very popular protein fold because it's so stable that it makes proteins resilient against heat stress and proteolysis. Getting the structure of a toxin allows us to classify the toxin and compare it with structurally similar toxins. Since APETx1 has structurally similarity with two other toxins BeKm1 and CnErg1, you can infer it would have a similar mechanism for blocking potassium channels. Chagot suggested that five key amino acids on APETx1 (i.e. Tyr 5, Tyr 32, Phe33, Lys8 and Lys 18) 'block' the Ether-a-gogo channels through electrostatic interactions.

Figure 2. 3-D structure for APETx1 (Source: Chagot et al, 2005)

In short, we can use a variety of biophysical techniques to get structural information for new toxins  and the structure provides insight into the function. Protein structure and function are different sides of the same coin. Next time, I will flip the coin by covering how we figure out function.

Citations:

Chagot, B., Diochot, S., Pimentel, C., Lazdunski, M. & Darbon, H. Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: A new fold for an HERG toxin. Proteins Struct. Funct. Genet. 59, 380–386 (2005).

Jouiaei, M. et al. Ancient venom systems: A review on cnidaria toxins. Toxins (Basel). 7, 2251–2271 (2015).

Norton, R. S. Structures of sea anemone toxins. Toxicon 54, 1075–1088 (2009).

Norton, R. S. & Pallaghy, P. K. The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36, 1573–1583 (1998).

Dauplais, M. et al. On the Convergent Evolution of Animal Toxins. J. Biol. Chem. 272, 4302–4309 (1997).

Belmonte, G. et al. Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles. BBA - Biomembr. 1192, 197–204 (1994).

Honma, T. & Shiomi, K. Review Article Peptide Toxins in Sea Anemones: Structural and Functional Aspects. Mar. Biotechnol. 8, 1–10 (2006).

Bernard Gilquin, Judith Racape, Anja Wrisch, Violeta Visan, Alain Lecoq, Stephan Grissmer, Andre´Menez, and S. G. Structure of the BgK-Kv1.1 Complex based on distance restraints identified by double mutant cycles: Molecular basis for convergent evolution of Kv1 channel blockers. J. Biol. Chem. 277, 37406–37413 (2002).

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Hunting for anemone toxins

Maybe some of you read my posts in Anemone toxins 101 and asked yourself, "How do people find anemone toxins and how do they know what these toxins do?"

Well, my friends, I am going to cover the arduous process of discovering and characterizing anemone toxins in Anemone Toxins 201. Once people figure out the sequence, structure and function of an anemone toxin, there are a lot of amazing you can do with anemone toxins. We can use anemone toxins to develop therapeutic proteins. For Ecologists, investigating the toxin profile of an anemone can provide a lot of information on its physiology, behaviour, predator-prey relationships, etc. As a Biophysics nerd, I was drawn to anemone toxins because they are a great tool for learning about ion channel biophysics.

In general, there are three main avenues for finding new anemone toxins: genomics, transcriptomics and proteomics. For genomics and transcriptomes, we extract the DNA and RNA respectively from the anemone's tissues for sequencing. For proteomic approach, you can just extract the protein and sequence it but there one major problem with this approach. The relative abundance of anemone toxins is much lower than all the other housekeeping proteins. A technique like MS/MS may not detect anemone toxins due to their lower relative abundance.

So we have to make our anemone smoothie and purify proteins with size exclusion or reverse phase chromatography, before trying to use protein sequencing for specific fractions. Or you use SDS PAGE for separating proteins by size. If you do MS/MS after isolating the anemone toxin, you have a much better chance of detecting and identifying the toxin.

Once we have our sequence, from the anemone transcriptome, genome or proteome we just dump it into BLAST. The gist of BLAST is that it uses multiple sequence alignments to see if there are other sequences (RNA, DNA or protein) in the database which are similar to our sequence. We kind of inferring what our new toxin is based on sequence similarity. The protein sequence dictates protein structure and function. So, if your new sequence is highly similar to a well-characterised anemone toxin, there is a decent chance that it will have a similar structure and function as the known toxin.

 Okay, so you have your BLAST output. What does it mean?

If you have read my 'Misadventures in undergrad research' post, you will know that the biggest challenge for me in my second project was figuring if my sequence was an actual anemone toxin.  

You know how to use this cool bioinformatics tool! Yay! Do you know how to interpret the data?

 Here are a few things to look out for:

 Conserved amino acids: Over time, as proteins evolve you’ll have some mutations. Sometimes, changing some minor amino acid is no big deal for the protein. However other amino acids are so important for folding, structure or function that the protein can't afford to lose them. These amino acids are conserved and you will see them being conserved in a variety of structurally or functionally similar proteins. For example, sometimes amino acids which are important for binding to potassium channel will be conserved from potassium channels toxins from different anemones.

Conserved cysteine residues: Cysteine residues are important for forming disulphide linkages and these linkages stabilise the entire 3-D structure. Sometimes ditching a disulphide linkage can impact the structure and function of a protein.

Conserved domains: Domains are functional or structural units which make up the protein. Just like conserved amino acids, conserved domains are important for function and structure. For anemone toxins, if you see that your protein has a conserved Kunitz domain, that tells you that your toxins probably acts as a serine protease inhibitor. It doesn't matter where you find your cytolysin, it could be a carpet anemone or a hellfire anemone, but almost all cytolysins will have a conserved cytotoxic domain because it required for penetrating the cell membrane.

Key residues which are different: Maybe the toxin is incredibly similar to a known toxin—expect for this one important amino acid. Sometimes minor differences in the amino acid sequence can have huge implications for the function. APETx1 and APETx2 are highly similar toxins from the same anemone, but they block completely different channels. APETx1 blocks potassium channels, whereas APETx2 block acid-sensing ion channels. It's all because of differences in a few key amino acids.

 Figure 1- The power of bioinformatics. Structure and MSA of two potassium channel toxins : ShK and BgK ( Jouiaei, M. et al, 2015) 

There is a minor problem with this approach because you can miss a lot of hidden gems. Some anemone toxins are so special, that you won't get a match with another anemone toxin. Maybe your hypothetical anemone toxin has similarity to spider or scorpion toxin. That doesn't mean that the anemone got bitten by a radioactive spider and now it makes spider toxins. Due to nature of evolution a lot of proteins with different functions in different organisms can similar motifs, binding sites and domains. Keep in mind that multiple sequence alignments only tell you what the toxin could be, not exactly what it is and what it does.For all you know the hypothetical protein does nothing.  It is important to synthesize the toxin and characterise the function.

You can go the microbial route and use E. coli to express your protein. Or you can do what I did as an undergrad: peptide synthesis. Peptide synthesis is more suitable synthesizing smaller toxins. Either way, you have to go through the process of purifying your protein with liquid chromatography. If you have the protein sequence, you can predict its isoelectric point, molecular weight and hydrophobicity. Once you have this information you can decide what's the best purification method e.g. ion exchange, size exclusion or reverse-phase.

So that's the gist of the first step to finding a novel toxin. Next time I will cover how to figure out the structure of anemone toxins.

Citations:

Anderluh, G. & Mac, P. Cytolytic peptide and protein toxins from sea anemones (Anthozoa : Actiniaria ). Toxicon 40, (2002).

Diochot, S., Baron, A., Rash, L. D., Deval, E., Escoubas, P., Scarzello, S., Lazdunski, M. (2004). A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. The EMBO journal, 23(7), 1516–1525. DOI:10.1038/sj.emboj.760017

Jouiaei, M. et al. Ancient venom systems: A review of cnidaria toxins. Toxins (Basel). 7, 2251–2271 (2015).

Norton, R. S. Sea Anemone Venom Peptides. Handbook of Biologically Active Peptides (Elsevier Inc., 2006). DOI:10.1016/B978-012369442-3/50056-8

Prentis, P. J., Pavasovic, A. & Norton, R. S. Sea anemones: Quiet achievers in the field of peptide toxins. Toxins (2018). doi:10.3390/toxins10010036

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Misadventures in undergrad research (Part III)

Finally, the sea anemone saga comes to a thrilling conclusion. I submitted my thesis, wrapped up my exams, and graduation is only two weeks away. After spending almost three years of my life obsessing over sea anemones and gushing about toxins, being the 'anemone girl' in my lab became a part of my identity. If I had to compare my research project trilogy with a film trilogy, then I would go with The Dark Knight Trilogy because in my final year project I fell into a giant pit and had to drag myself out.

My FYP was a sequel to my second research project. I had identified potential potassium channel toxins in my second project, so the next was step was synthesizing and purifying the toxins for structural and functional characterization. Throughout my FYP, the actual approach and direction for my project kept changing because I was struggling to synthesize my toxins. The original idea was: make the toxin and find the disulfide linkages. That idea was transformed into: keep trying to make the toxin while predicting the structure and do CD once the toxin was produced. That idea was changed to: just predict everything and hope that you get something at the end.

After my first synthesis, I spent my lot of time trying to purify the toxin but then one my lab mates pointed out the horrifying possibility the peptide with the right mass wasn't even the crude sample. So I had to go back to the drawing board and figure out why my peptide wasn't there. A lot of times as an undergrad, you can fall into the trap of clinging onto a technique or approach while, believing that if you try just one more time, it could finally work. That's how you can end up wasting time pursuing a dead end. You have to be honest with yourself about your project and admit when something isn't working, even if you poured hours into your experiments and thesis. I have lost track of the number of times I scrapped things from my thesis.

For some bizarre reason, I couldn't synthesize my peptide. Even when we sent the sequences to a company which specializes in peptide synthesis, they were also having problems making the damn thing. Obviously, my toxins were cursed. I tried multiple strategies to synthesize my peptide, and through the process of trial and error; I ended up gaining a more in-depth understanding of peptide synthesis. Had I gotten my peptide in the first try I don't think I would have learned as much.

Half through the second act of my project, I realized there wasn't enough time to find the disulfide linkages experimentally. That's when I ended up expanding the Bioinformatics section of my thesis. I had taken only taken one Bioinformatics course as an undergrad, so it was kind of out of my comfort zone. By the time I had to start writing the first draft, I had lost track of the plot of my movie because I had tried so many different things. I just sat down one day and made a flow chart of all the experiments I did. There it was, a clear narrative which I could use to structure my thesis. Storytelling has always been a tool that I have used to make sense of my life and identity, but this is the first time I used storytelling to make sense of a research project.

Every undergrad and grad student's worst nightmare is that their research project isn't going anywhere. Sometimes you'll spiral into an existential crisis as you stare at mass spectra data and question your own sanity and competence. I think it is easy to fall into the trap of tying your sense of self-esteem to your results. After that, every single failure slowly destroys your confidence, motivation, and passion for the project. Once you lose your motivation, it becomes even harder to solve problems. The phrase: "What am I going to put in my thesis?" haunted me through my project. Here's the thing,  even if your results are less than ideal or you weren't able to synthesize something, you still have a story to tell. You can use that story to convey what you have learned and what makes the 'character' or 'theme' that focusing on in your project special.

One of the main challenges of doing research is that you have to deal with failure and uncertainty—a lot. We strive to be as objective and rational as possible, but ultimately, we are flawed humans who are limited by our perception, biases, and experiences. Even if you executed every experiment perfectly, there are factors beyond your control, which impact the results.  Research papers are cyclical in a way because they start out by tackling a gap in our knowledge and towards the end, they point out another gap in our understanding in the future work section. There is an existential element to doing research. We are living an indifferent and complex universe, without a cosmic mentor figure who can give us all of the answers.

All we can do is try our best to fill in the gaps.

[lifesciencepotluck]

New Kids on the Block

The reason why I get so excited and giddy when it comes to anemone toxins is that people keep discovering new types of anemone toxins. Unlike snake toxins, the ocean of anemone toxins hasn’t been explored as much. It's only in the early 1970s that people started identifying and studying anemone neurotoxins. The field of anemone toxins is like as young child which is still growing and developing. Right now, we don't have that many toxins in the database. We have information for the 3D structure, functional characterization, structure-function relationships and pharmacological properties for very few toxins. There are more than 900 species of anemones, and currently, the venom from less than 10% of species that have been studied. So, the field of anemone toxins is underrated and understudied.

We need more people.

Like seriously, we really need more people.

The types of toxins that I talked about in my previous posts were discovered early on the in the history of anemone toxins. However, the types of toxins I am going to talk about in this post have been found pretty recently. Hence the title, New Kids on the Block. 

ASIC inhibitors 

Acid-sensing ion channels (ASICs) are proton-gated sodium channels. These ion channels are found in the peripheral neuronal system, and they play a part in pain modulation. There are many types of ASIC because there are six different kinds of subunits which can be arranged a bunch of different combinations. ASICs are therapeutic targets due to the wide variety of roles they play in the human body (e.g., pain modulation, mood, and even memory). Researchers have managed to isolate anemone toxins which target acid-sensing ion channels. APETx2 is a ASIC inhibitor which has 42 amino acids and two disulfide bridges. It has some sequence similarity with Type III potassium channels toxins such as BDS I. However; it inhibits ASIC3 instead of potassium channels. Other examples of anemone toxins which inhibit ASICs include PhcrTx1 from Phymanthus crucifer and π-AnmTX Ugr 9a-1 from Urticina grebelnyi.

Trpv1 inhibitors

TRPV1s are non-selective cation channels. Potassium channels will only K+ ions to pass through, but TRPV1 are less picky when it comes to the type of positive ions which can pass through. TRPV1s are found in the peripheral and neuronal system. Sometimes you'll find TRPV1s in the central nervous system. TRPV1s play a role in responding to a variety of chemical (acidic conditions) and physical stimuli. Some TRPV1 receptors which have been studied in more depth, play a part in detecting and regulating body temperature. These channels initiate a response when there is inflammation, which makes them a therapeutic target. τ-SHTX-Hcr2b (APHC1) was the first peptidic TRPV1 inhibitor found in anemone venom, and it was isolated from Heteractis crispa. There could be other species of anemone out there which could provide us with even more TRPV1 inhibitors. These inhibitors could be used to develop analgesic drugs. 

Small Cysteine-Rich Peptides (SCRiPs)

SCRiPs are a recently discovered group of neurotoxins which have been found in reef-building corals such as Acropora millepora and some species of sea anemones such as Anemonia viridis and Metridium senile. When they injected SCRiPs from Acropora millepora into zebrafish larvae, it caused severe paralysis. SCRiPs are also found in certain species of plants. These SCRiPs have antimicrobial properties and they protect plants from pathogens. So SCRiPs can play different roles in different organisms. Unlike sodium channels toxins, the functional aspects of SCRiPs in anemone venom is still a bit of mystery.

Of course, there are recently discovered anemone toxins which don't fall into any of the categories that I have mentioned in my posts. I keep mentioning Acrorhagin I and Acrorhagin I, because they are the like weird sisters of anemone toxins. These toxins don’t have homology with anemone toxins. However, they have some sequence similarity with spider toxins and conotoxins (these are toxins found in cute little cone snails). Here’s what we know about the function of Acrorhagin I/II—they are neurotoxins. That’s it. We don’t know what kind of channel or receptor they target. 

Anemone venom is like a magical and massive candy shop for toxins. You will find a wide variety of weird peptides that try to exploit the physiological features of other organisms. 

Well, that’s the end of Anemone Toxins 101. I have provided a basic introduction to anemone toxins for anyone else who is interested in this weirdly specific field, that I love. 

Citations

Honma, T. et al. Novel peptide toxins from acrorhagi , aggressive organs of the sea anemone Actinia equina. Toxicon 46, 768–774 (2005).

Jouiaei, M. et al. Ancient venom systems: A review on cnidaria toxins. Toxins (Basel). 7, 2251–2271 (2015).

Logashina, Y. A. et al. New disulfide-stabilized fold provides sea anemone peptide to exhibit both antimicrobial and TRPA1 potentiating properties. Toxins (Basel). 9, (2017).

Prentis, P. J., Pavasovic, A. & Norton, R. S. Sea anemones: Quiet achievers in the field of peptide toxins. Toxins (Basel). 10, (2018) 

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Evil enzymes and biochemical bombs

Now that I have covered the ripper toxins and neurotoxins, it is time to take about enzymes. Ah, enzymes. They help digest food, catalyze important reactions, destroy pathetic prey—yes, anemones and other venomous animals have somehow managed to use enzymes to kill things. In anemones, there are two major kinds of enzymes which are classified as toxins: phospholipases A2 (PLA2s) and metalloproteases. Aside from anemones, metalloproteases are also found in as snakes and ticks. These diabolical toxins cause hemorrhage and necrosis by destroying the extracellular matrix (ECM). The ECM is a vital piece of biological architecture which provides structural and biochemical support for cells. It is composed of collagen, enzymes, and glycoproteins. All of these lovely proteins get degraded by the anemone’s merciless metalloproteases.

 Not only do anemone wreck cells with ripper toxins and use neurotoxins to attack ion channels, but they also destroy the support structure for cells. Why not attack cell membranes and myelin with phospholipases A2 (PLA2s)?

PLA2 toxins catalyze the hydrolysis of 2-acyl ester bonds glycerophospholipids. Yes, even the cell membranes aren’t safe from an anemone’s venom. The funny thing is that your pancreas uses PLA2 to digest that sandwich you had for breakfast, but a lot of venomous animals use PLA2 to kill things. PLA2 toxins could serve a bunch of different roles like defense, immobilization, and digestion. The literature suggests that PLA2 could play multiple roles which makes it a bit to tricky study the structure-function relationship of these toxins.

Figure 1: Predicted strcuture of UcPLA2. The toxin was isolated from the starlet sea anemone (Nematostella vectensis) [Source: Razpotnik, A. et al, 2010]

Just like potassium channel toxins are classified based on structure, PLA2 toxins are also classified based on structural features into four categories: phospholipase A1, phospholipase B, phospholipase C, and phospholipase D. There is a secreted family of PLA2 which need calcium ions to be active. There are certain types of PLA2’s which are found in the cytosol. They can depend on calcium ions for activity, or they can be strong independent enzymes.

Aside from a dash of potent peptides anemone venom has other intresting ingredients. 

You see, almost every component in the anemone’s venom plays a part in making sure that the prey or predator dies a slow and excruciatingly painful death. In some cases, anemone venom can also have a pinch of 5-hydroxytryptamine (5-HT, serotonin), histamine, bunodosine, and caissarone. 5-HT enhances vasodilation which enhances the impact of all the other toxins and histamine is excellent for causing sharp pain within victims. Bunodosine is an N-acylamino acid which has analgesic activity, and it targets serotonin receptors. Caissarone is a purine derivative which acts as an antagonist against adenosine receptors. The antagonistic adventures of this lovely ligand involve blocking or dampening the activity of adenosine receptors. You know what else acts as an antagonist against adenosine receptors?

Caffeine.

Figure 2:  Bunodosoma cangicum (left). Chemical structure of bunodosine (right).  [Soruce: Zaharenko, A. J. et al, 2011]

Most of my blog posts in Anemone toxins 101 focus on the toxins, but there are other exciting ligands in anemone venom which aren’t peptides. Bunodosine and caissarone could be developed into some pretty potent marine drugs. Even though I am fascinated with the biomedical applications of anemone toxins specifically, I think other components of the venom shouldn't be glossed over.

Next time, I will talk about the—New Kids on the Block.

Citations

Jouiaei, M., Yanagihara, A. A., Madio, B., Nevalainen, T. J., Alewood, P. F., & Fry, B. G. (2015). Ancient venom systems: A review on cnidaria toxins. Toxins, 7(6), 2251–2271. http://doi.org/10.3390/toxins7062251

Lehtonen, J. Y., & Kinnunen, P. K. (1995). Phospholipase A2 as a mechanosensor. Biophysical journal, 68(5), 1888-94.

Razpotnik, A. et al. A new phospholipase A2 isolated from the sea anemone Urticina crassicornis - Its primary structure and phylogenetic classification. FEBS J. 277, 2641–2653 (2010).

Zaharenko, A. J. et al. Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum. J. Nat. Prod. 74, 378–382 (2011).

[lifesciencepotluck]

Potassium channel toxins

I constantly joke about how carpet anemones are like the McDonalds’s of anemone species where I live because you can practically find them anywhere. However, they play a pivotal part in the history of anemone toxins. The first potassium channel blockers were found in Stichodactyla helianthus, and the genus Stichodactyla has provided a bunch of toxins that were engineered into really potent and useful drugs. SHK is a classic example of taking an anemone toxin, doing a bit of protein engineering and creating medicine out of poison. Compared to sodium channels, there are WAY more types of potassium channels out there. Which could explain why there is so much diversity in the targets, sequences, and structures of potassium channel toxins. Some of these toxins work as pore blockers like Aek and BgK. Some of these toxins are potassium channel modifiers like BDS I. Sometimes you get a 2-in-1 deal since some potassium channel toxins also work as serine protease inhibitors or they can target calcium channels.

Figure 1: 3D structure of ShK, the toxin that has been developed into some pretty great analogs and drugs. (Source: PDB) 

Of course, as always…there is a classification system. This classification system classifies anemone toxins based on structural similarities.  It’s not like, group 1 potassium channel toxins target Kv1 channels. Nope, the toxins are classified based on sequence length and number of disulfide linkages.  In some groups, the potassium channel toxins have structural similarities like a Defensin domain, but they end up targeting completely different types of potassium channels, and they have different affinities for certain kinds of channels.

Type I potassium channel toxins have 35-37 amino acids and three disulfide bridges. They tend to block currents running through KV1 subunits, and they also mess with K(Ca) channels. Most of the times the toxin in this clique are potassium channel blockers; this means that they go inside the channel and physically block the pore. How do they do this? Well, they have a secret weapon known as…the lysine-tyrosine dyad. This dyad in conserved in not only anemone potassium channel blockers, but it also conserved in toxins from other animals such as scorpions and cone snails. BgK is a classic example of potassium channel blocker. 

Figure 2: 3D structure of BgK (Source: PDB) 

Type II potassium channel toxins have 58–59 amino acids and three disulfide bridges. They tend to have homology with Kunitz-type inhibitors, which means that they act as serine proteases inhibitors. You must be wondering, why does an anemone need that? The main advantage of having a Kunitz type toxin in your arsenal is that you can stop the proteases inside the prey from digesting your toxins. Our type II potassium channel toxins end up playing two parts. They play a role in protecting the toxins in the anemone’s venom against the host’s defenses, and they also help paralyze the target.

Type III potassium channel toxins have 41–42 amino acids and three disulfide bridges. Unlike our type I friends, these toxins are channel modifiers which means that they mess with the gating mechanism of potassium channels. Toxins like BDS I and BDS II target KV3 subunits or ERG (ether-a-go-go, KV11.1) channels. Cardiac cells have ERG channels and toxins like BDS I could be used to develop drugs to target heart disease. These toxins may have gotten the potassium channel toxins label, but they actually have structural similarities with sodium channel toxins. The literature suggests that these toxins evolved from sodium channel toxins. APETx2 has similarity with other type III potassium channel toxins, but it actually targets acid-sensing ion channels they’re basically; proton-gated sodium channels which play a part in pain modulation. See, these are the kinds of plot twists you can expect in the field of anemone toxins. Something looks and talks like a potassium channel toxin but ends up acting like an ASIC inhibitor instead.

Figure 3: 3D structure of BDS I (Source: PDB) 

Type IV and Type V are the new categories for anemone potassium channel toxins that don’t fit in the three first three categories.  Type IV toxins get their own category because it only has two disulfide bridges instead of three disulfide bridges like the other anemone toxins.

I have to be honest with you; potassium channel toxins are my favorite type of anemone toxin. My undergraduate research focuses on potassium channel toxins. So, I may have developed, a bit of an obsession a fascination with them. The best part is that people keep finding new potassium channel toxins that are completely different from well-known potassium channel toxins in terms of sequence, structure, and function. Someday we could find anemone toxins to target each type of potassium channel. Maybe there are chlorine channel toxins or magnesium channel toxins out there. Who knows? Right now, anything is possible in the field of anemone toxins, and that’s absolutely amazing.

Citations

Frazão, B., Vasconcelos, V., & Antunes, A. (2012). Sea anemone (cnidaria, anthozoa, actiniaria) toxins: An overview. Marine Drugs, 10(8), 1812–1851. http://doi.org/10.3390/md10081812

Jouiaei, M., Yanagihara, A. A., Madio, B., Nevalainen, T. J., Alewood, P. F., & Fry, B. G. (2015). Ancient venom systems: A review on cnidaria toxins. Toxins, 7(6), 2251–2271. http://doi.org/10.3390/toxins7062251

Aspects, F. (2006). Review Article Peptide Toxins in Sea Anemones: Structural and, 8, 1–10. http://doi.org/10.1007/s10126-005-5093-2

Norton, R. S. (2009). Structures of sea anemone toxins. Toxicon, 54(8), 1075–1088. http://doi.org/10.1016/j.toxicon.2009.02.035

[lifesciencepotluck]

Sodium channel toxins

Neurotoxins (e.g. your potassium and sodium channel toxins) are pretty well known and well characterized for sea anemones. Which isn’t surprising since the first anemone toxins that were identified in 1975 were neurotoxins. They play a vital part in paralyzing prey or predator. They tend to be on the smaller side in terms of molecular weight. In general, there are two types of neurotoxins. There are the ones, which physically go inside the pore of the ion channel and block it by hogging space like an inconsiderate moocher. Then there are the ones that bind to the channel to screw up the structure of the ion channel. The channel modifiers can extend the action potential of both the excited and non-excited neurons, which is why they are such fantastic tools for studying how ion channels work.

A neurotoxins general plan of attack.

Step 1: Either modify an ion channel or block the pore.

Step 2: Cells become hyperactive and release a boat load of neurotransmitters.

Step 3: Sadistically watch the horrifying and uncontrollable flood of neurotransmitters cause paralysis.

Now, you have to keep in mind, that anemone toxin doesn’t have all year to prance around the prey’s body to disrupt ion channels. Overtime it will get degraded by the host’s own enzymes which is one of the reasons why each toxin has its own specific target (e.g. some potassium channel toxins will only target ion channels found in the heart cells or skeletal cells). The reason why we keep mining for novel potassium or sodium channel toxins because they can have affinities for different types of channels or they can have a different level of efficacy. I’ll start out with sodium channel toxins and cover potassium channel toxins in my next post.

Figure 1: ATX-II is sodium channel toxin from Anemonia sulcata which spefically binds to  NAv1.1 and Nav1.2 channels 

Sodium channel toxins tend to make up a large part of the venom. Majority of sodium channel toxins (AKA our Type I and Type II toxins) bind to site 3 of the sodium channel in order to modify the channel. In reality there are seven possible binding sites for sodium channel toxins to exploit in order to screw up the sodium channels. There is a chance that there are undiscovered anemone toxins which could bind to these sites. Site 3 is in the loop S3–S4 of the fourth domain in the sodium channel toxin. It's implied that the electrostatic interaction between a cluster of basic amino acids in the toxin and site 3, causes a change in structure that delays the deactivation of our sodium channel. Now, this means you have an insane amount of sodium ions flooding into the channel, because it can't shut down. It's the ion channel equivalent of an already sugar high hyperactive kid gorging on even more sugar and soda. That’s where we end up with our neurotransmitter overdose and painful paralysis.

Figure 2: Binding site for most sodium channel toxins (Type I and Type 2) within sodium channel. 

Currently there are four types of sodium channel toxins. These toxins are grouped based on the tertiary or secondary structural features as well as the disulphide linkages and sequence similarity. Type 1 and 2 have around 46-51 amino acids and a key structural feature is the anti-parallel beta-sheet with a flexible loop (also known as the arginine loop). They tend to have conversed cysteine residues. So, if you look at the multiple sequence alignment for Type I and Type II toxins, then the cysteines at the same position even though all the other amino acids can differ. That’s how you know that these residues are a big deal because the natural selection fairy basically said, ‘Hey there these, residues play a big part in your structure and function. Don't lose them over many of years of evolution— you idiot.’ They also have basic residues like lysine or arginine at the C terminus which they tend to help with the binding and modification process. 

Type III toxins are smaller since they only have 27-32 amino acids and they don’t have any fancy beta-sheets or alpha helices. They just have rigid beta and gamma turns. Even though the sequences tend to be similar for this group they don't share any structural scaffolds in common. So, the moral of the story for all the protein lovers out there is that, just because two peptides have completely different sequences doesn't mean that they can't have structural similarity or if two sequences are actually really similar the structural features can be completely different. 

Type IV is the outcast group for sodium toxins don't really fit into any categories. In older papers, there were only Type I and Type II toxins. Any toxins that didn't fit into these two categories were dumped into the orphan NaTx clade, which is kinda sad. C’mon even the name of the clade is depressing as hell. It’s Oliver Twist but with sodium channel toxins.

But I know that someday there will be a happy ending to the story because a bunch of people obsessed with anemone toxins will find and characterize new sodium channel toxins. And maybe, just maybe every sodium channel toxin will actually belong somewhere instead of being dumped into ‘that’ group. 

Citations

Frazão, B., Vasconcelos, V., & Antunes, A. (2012). Sea anemone (cnidaria, anthozoa, actiniaria) toxins: An overview. Marine Drugs, 10(8), 1812–1851. http://doi.org/10.3390/md10081812

Jouiaei, M., Yanagihara, A. A., Madio, B., Nevalainen, T. J., Alewood, P. F., & Fry, B. G. (2015). Ancient venom systems: A review on cnidaria toxins. Toxins, 7(6), 2251–2271. http://doi.org/10.3390/toxins7062251

Aspects, F. (2006). Review Article Peptide Toxins in Sea Anemones: Structural and, 8, 1–10. http://doi.org/10.1007/s10126-005-5093-2

Norton, R. S. (2009). Structures of sea anemone toxins. Toxicon, 54(8), 1075–1088. http://doi.org/10.1016/j.toxicon.2009.02.035

[lifesciencepotluck]

Cytolysins and Actinoporins

It’s kinda funny that it took me five posts before I started writing about actual anemone toxins, but I thought proving context was important before I dive into the really juicy bits. Compared to other cnidarians (e.g. Jellyfish), anemones have more variety in the types of toxins they use.

Here is a short (but by no means comprehensive) list of all the goodies in anemone venom: cytolysins, actinoporins, potassium channel toxins, sodium channel toxins, phosolipases, metalloproteases, serine protease inhibitors/Kunitz peptides, ASIC (acid sensing ion channel) inhibitors, TRPV1 inhibitors and small cysteine rich peptides (SCRiPs). In this post, I’ll focus on the ‘ripper toxins’ AKA your cytolysins and actinoporins.

Figure 1: "Ripper” toxins in action

Almost every cnidarian species has a “ripper toxin” or a psychotic molecular chainsaw that rips through the cell membrane and causes water to flood into the cell, until it swells and bursts due to osmotic pressure. There are two contradictory parts to our pore forming toxin: one part is hydrophilic (or water loving/water soluble) and it binds to the receptors of our poor cell. The second part is the membrane bound structure which is more hydrophobic since it plays a part in pore formation. There are two main categories of pore forming toxins: actinoporins and cytolysins.

Actinoporins (~20 kDa) have a really simple structure which really shows when you realize that they don't even have any disulphide bridges. Some actinoporins will only bind to sphingomyelin while others can bind to phosphatidylcholine in membranes (E.g. Sticholysin II). Actinoporins plan of attack involves using their aromatic loops to bind to the membrane, aligning themselves in the perfect position for cellular murder, oligomerising and then shoving the N-terminus of their alpha helix into the membrane like a tiny drill. Some studies suggest that the amphiphilic alpha helix actually detaches from the main body of toxin before drilling into the membrane. Since actinoporins can causes cardiovascular and respiratory problems, you can usually use a hemolytic assay to experimentally figure out if you have actinoporins or cytolysins in samples.

Figure 2: A diagram of pore forming toxin with our two key components

One of the appeals of studying anemone toxins, is using toxins to mess with ion channels or cell membranes in order to get an understanding of how cell membranes and ion channels work. A lot of times in Biology we learn about how things work by screwing up specific parts of proteins, cells, genomes etc. Cytolysins have been used in studies to study cell membrane dynamics and structure. Since biologists are a bunch of pedantic nerds who have to classify everything they categorize cytolysins based on molecular weights and other commonalities.

Figure 3: 3D stcrure of EQUINATOXIN II

Type I, cytolysins tend to have a molecular weight of 5–8 kDa peptides and they tend to form pores in phosphatidylcholine containing membranes. They have antihistamine activity (plays a part in the nervous and cardiovascular system). Some type I cytolysins have no cysteine residues and they tend to have high as hell isoelectric points.

Type II (~20 kDa) cytolysins are really common and well-studied. They tend to target phospholipid domains, get inhibited by sphingomyelin and they oligomerise to form pores. One of the key structural features is the cation selective hydrophobic core which can cause haemolysis.

Type III (30–40 kDa) cytolysins can have PLA (phosolipases A) activity in some cases. 

The largest type of cytolysins are Type IV cytolysins (~80 kDa) and they are thiol-activated. 

Why do you need to know about these types of cytolysins? 

FOR FUN! And also, because, knowing the size of your toxin helps with isolation and identification of specific toxins. For example, you get your crude anemone venom (through some magical miracle) and do SDS PAGE to get a rough venom profile. If you know the approximate sizes you can guess what kind of toxins are in the anemone’s venom.

This post is a brief introduction to actinoporins and cytolysins. I could showcase specific cytolysins or dive into the structure-function relationships in future posts. (Even more foreshadowing….)

Citations

 Norton, R. S. (2009). Structures of sea anemone toxins. Toxicon, 54(8), 1075–1088. http://doi.org/10.1016/j.toxicon.2009.02.035

Norton, R. S. (2006). Sea Anemone Venom Peptides. Handbook of Biologically Active Peptides. Elsevier Inc. http://doi.org/10.1016/B978-012369442-3/50056-8

Jouiaei, M., Yanagihara, A. A., Madio, B., Nevalainen, T. J., Alewood, P. F., & Fry, B. G. (2015). Ancient venom systems: A review on cnidaria toxins. Toxins, 7(6), 2251–2271. http://doi.org/10.3390/toxins7062251

Rojko, N., Dalla Serra, M., Maček, P. & Anderluh, G. Pore formation by actinoporins, cytolysins from sea anemones. Biochim. Biophys. Acta - Biomembr.1858, 446–456 (2016).

[lifesciencepotluck]

Venom extraction and you!

For most venous animals like snakes and bees, once you find the venom gland you have your gold mine of toxins. However, because Cnidarians have such a weird way of storing toxins, venom extraction with sea anemones is a bit more complicated. Trying to extract the venom from a sea anemone is like trying to pool a thousand microscopic drops of gold together in the hopes of having enough venom to study the actual toxins. Which is why a lot of desperate scientists with a thirst for anemone poison have come up with some pretty creative ways of extracting venom. I am going to put some of these techniques into two board categories: ones where the anemone lives and the ones where you have to be an anemone assassin.  

Approach 1: Pray that you hit the right magic button and the anemone gods give you their sweet venom

These techniques involve keeping the anemone alive and annoying the hell out of them until they give you a bit of venom.  One approach involves poking the anemones throat with a rod, until exports out a bit mucus from its mouth. You can get your anemone mucus by squeezing the column until the mucus oozes out. You can also try shocking your anemone into firing cnidae by tossing it into distilled water or a sodium citrate solution. Doing either of this things causes the anemone stress due to the change is osmotic pressure, but if your species of anemone is too sensitive it will die. One of the funniest methods that I read about involved making the anemone your cow. Basically, you stuff the anemone with shrimp or crab (it depends on the typical diet for that species of anemone) and then you starve it for a few months. The ‘milking stage’ involves squeezing the stressed-out anemone, for you guessed it—the mucus. Maybe you have secret sadistic urges. Well, you can live out your fantasies by putting an electrode inside of the anemone’s gastrovascular cavity and giving it a mild shock. Yes, this is an actual venom extraction technique, even though it sounds a cross between a marine biology and a medieval torture device.

Most of these methods involve stressing or shocking the anemone because sudden stress acts as a stimulus for the cnidae to fire and release some of the venom. The type of technique you use for the living specimen really depends on the anatomy and physiology of your species of anemone. Some of these methods are too harsh for certain species of anemones. Most of these methods can give you very good purity for the venom but the yield tends to be on the lower side. Maybe you are tired of nagging your anemone for venom, well this where the second approach comes in.

Approach 2: Throw the sucker in a blender and make a sea anemone smoothie

In general, this approach involves homogenizing your anemone and then purifying the crude extract with a combination of different chromatography techniques (like Size Exclusion and Reverse phase) or using a gel to separate the proteins (E.g. SDS PAGE). It is probably one of the most commonly used approaches due to how convenient and fast it is. It is also, one of the older venom extraction techniques. The advent of reliable purification techniques like gel filtration was pivotal for not only getting partially purified venom extracts in the 1960’s but also for isolating and identifying one of the first anemone neurotoxins toxins from Anemonia viridis in 1975. Some studies homogenise the anemone by literally throwing the anemone in a blender, other techniques use cycles of thawing and freezing the anemone for homogenisation or a simple magnetic stirring to break up those tantalising tentacles.

After that you have separate your toxin peptides with size exclusion chromatography or reverse phase or anion exchange chromatography. Once you figure out which fractions have the toxin (you can use a crab assay where you poison adorable crabs with a sample from the fraction and see which fractions ‘hurts’ them) you have to purify it again with another round of reverse phase so you have pure enough sample for protein sequencing. The problem with this approach is that you will get a high yield but the purify of the crude venom extract will be pathetic (hence the billion purification steps).

One of more the recent modifications to this approach involves isolating the nematocysts (stinging cells) from the anemone and extracting the venom from the cnidae. Basically, the author suggests taking the tentacles or acontia and the using a salt gradient to separate the different types of nematocysts based on size (with spirocysts being adorably small and p-mastigophores being huge as hell).  Then you check what kind of nematocyst you have with a microscope and you can use a sodium citrate solution to cause the cnidae to swell up and fire. That’s how you get your venom at the end and this method allows you to get a purer sample. 

I may sound a bit frustrated in this post but it's because extracting the venom from cnidarians is a genuine challenge. I love how people have come up with a variety of weird but inventive ways of extracting venom from anemones. Hopefully, the development of new technologies makes it easier to extract venom from cnidarians in the future.

Citations

Greenwood, P. G., Balboni, I. M., & Lohmann, C. (2003). A sea anemone’s environment  affects discharge of its isolated nematocysts. Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology, 134(2), 275–281. http://doi.org/10.1016/S1095-6433(02)00262-3

Eno, A. E., Konya, R. S., & Ibu, J. O. (1998). Biological properties of a venom extract from the sea anemone, Bunodosoma cavernata. Toxicon, 36(12), 2013–2020. http://doi.org/10.1016/S0041-0101(98)00003-8 

Marino, A., Valveri, V., Muià, C., Crupi, R., Rizzo, G., Musci, G., & La Spada, G. (2004). Cytotoxicity of the nematocyst venom from the sea anemone Aiptasia mutabilis. Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 139(4), 295–301. http://doi.org/10.1016/j.cca.2004.12.008

Ramkumar, S., S, A. S., & Venkateshvaran, K. (2012). Bioactivity of venom extracted from the sea anemone Anthopleura asiatica (Cnidaria: Anthozoa): Toxicity and Histopathological studies. International Journal of Fisheries and Aquaculture, 4(4), 71–76. http://doi.org/10.5897/IJFA11.019

MALPEZZI, E. L. A., FREITAS, J. C., MURAMOTO, K., & KAMIYA, H. (1993). Characterization of Peptides in Sea Anemone Venom Collected By a Novel Procedure. Toxicon, 31(7), 853–8

Romero, L., Marcussi, S., Marchi-Salvador, D. P., Silva, F. P., Fuly, A. L., Stábeli, R. G., … Soares, A. M. (2010). Enzymatic and structural characterization of a basic phospholipase A 2 from the sea anemone Condylactis gigantea. Biochimie, 92(8), 1063–1071. http://doi.org/10.1016/j.biochi.2010.05.007

 Sánchez-Rodríguez, J., Zugatsi, A., Santamarı, A., Galva-Arzate, S., & Segura-Puertas, L. (2006). Isolation, partial purification and characterization of active polypeptide from the sea anemone Bartholomea annulata. Basic & Clinical …, 99, 116–121. http://doi.org/10.1111/j.1742-7843.2006.pto_428.x 

[lifesciencepotluck]

Size Exclusion Chromatography

Compared to Reverse-phase, Size Exclusion Chromatography (SEC) or Gel Filtration is a bit more straightforward. As stated in the name it separates proteins based on size (or hydrodynamic volume). Imagine that you want to get the molecular weight of your peptide but there is a butt load of salt which will interfere with mass spectrometry since it relies on the mass: charge ratio.

Gel Filtration is a great for removing salt from your sample. Since you don't use harsh reagents you don't lose the biological activity of your protein. As usual there is a stationary phase and mobile phase. The stationary phase is made of particles or beads with pores of a specific size. The mobile phase is the buffer flowing through the particles and it carries the salts, proteins and everything else in the sample that you apply to the column. The smaller particles (or your tiny salt ions) will flow into the beads and get retained but the larger molecules (the proteins that you actually care about) will flow past the beads and get eluted. It's harder for the column to retain larger proteins so those proteins get eluted at a smaller buffer volume or retention time. The smaller proteins will get eluted with a larger volume of buffer or higher retention time. Since there is a linear relationship between the molecular weight of a protein and the elution volume, so you can use size exclusion to get a rough estimate of protein’s molecular weight.

Figure 1: Chromatogram with elution volumes for proteins of different sizes.  

Figure 2: This is a bead of particle found in a typicla gel filtration column. The bead has tiny pores where small and adorabe solutes (like salt) can actually flow through the resin. Most of the proteins are too big to pass through these tiny pores so they actually flow through the column a lot faster, that’s why you need to less of your buffer to elute larger proteins.

Figure 3: Molecular weight vs Kav (or binding affinity of the protein for column)

A short list of boring but important jargon:

Void volume (V0): This the elution volume of stuff that doesn’t enter the pores or interact with the chromatography resin. These solutes just pass by the particles in the packed bed.

Internal volume (Vi): The volume of the pores within the gel. 

Total volume (Vt): This is the total volume of your column. (It is usually the sum of internal and void volume)

Elution Volume (Ve): The volume of the buffer needed to elute a specific solute. 

Stuff you need to keep in mind

Type of column and pore size: A longer column can give you a better separation of proteins, but it is the improvement in resolution is only proportional to the square root of the column length. Connecting columns with different pore sizes can help separate proteins with a wider range of molecular weights. You have to select your column based on the size of the target protein. Some columns can separate proteins with wide range of sizes (100-10 Kda) others are more suitable for separating smaller proteins (<10 Kda). The uniformity of the pore size is just important as pore size. Generally, if the pore size of the particles is larger you get poorer resolution.

Flow rate: Usually a slower flow rate leads to leads to better resolution or separation of proteins. However, one of the biggest drawbacks of size exclusion is the ridiculously long run time, since the flow rate tends to be slower than other forms like HPLC like Reverse-phase. The typical flow rate is usually between 0.5 ml/min and 1 ml/min. 

Another time-consuming aspect of SEC is the equilibrating the column before use and washing it after use. Sometimes the more hydrophobic proteins end up aggregating and sticking to the particles in the column (like an annoying piece of gum) and you have to use really hydrophobic reagents to wash those proteins out (like Acetonitrile).

Reagents: The buffer you use doesn't affect resolution that much but you want to pick a buffer with low ionic strength and ideal for storing proteins (e.g. ammonium bicarbonate buffer). Usually the beads in the column are silica based so they are a bit hydrophobic and have a weak negative charge.  So, what can happen is that proteins can end up getting retained by the anionic and hydrophobic beads, which gets in the way of eluting and collecting the proteins. Since you don't want any kind of ionic or hydrophobic interactions between the column and the solutes/proteins, adding a bit of salt to the solvent or mobile phase reduces these interactions and makes it easier to elute your proteins.

Another thing to keep in mind is that the shape of the protein affects how it moves through the column. A more rod like protein can move more easily than a more globular protein. BUT we want the column to separate the proteins based soley on size, so a tiny bit of a denaturing reagent (like SDS and guanidine hydrochloride) is added to the buffer. All of the proteins end up with a rod like shape and they are only separated based on size. 

Primary sources

Barth, H. G., Jackson, C. & Boyes, B. E. Size Exclusion Chromatography. Anal. Chem. 66, 595–620 (1994).

Nagy, K. & Vékey, K. Separation methods. Med. Appl. Mass Spectrom. 61–92 (2008). doi:10.1016/B978-044451980-1.50007-0

Recommended readings

GE Healthcare. Size exclusion chromatography: Principles and Methods. GE Heal. Handbooks 139 (2012).

GE Healthcare. Size exclusion chromatography columns and media Selection guide. 1–10 (2016).

[lifesciencepotluck]

Undergrad Research Tips: Literature Review

When you start a brand new research project you look at the vast expanse of possibilities in front of you and think, "Where the hell am I suppose to start?" There is where the literature review comes in. You have to do a butt load of reading so you actually have a chance to understand the research you're conducting. If you're lucky enough your prof will give you a list of recommended readings, but I urge you to go beyond. Start hoarding important studies, manuals and textbooks like a paranoid squirrel preparing for a neverending winter.

1. Find a solid review study that provides a good introduction to your field and gives a bit of context. If you look a scientific journal they have a variety of pieces ranging from research papers to editorials to opinion pieces to review studies. A review study is a kind of like the voice-over narrator in a trailer. They're like: In a world with anemone toxins...blah blah blah. For me when I was started learning about anemone toxins, Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview was a great introduction to the field. Once you get the basics down you can look at specific things in that field in more detail (e.g. later on, you could find more studies on potassium channel toxins). You can also go the citations section of a review study to find even more studies that relevant to specific concepts.

2. Get all of your manuals and protocols! If you are using a lab technique that's new to you like peptide synthesis or FRET get your hands on the manual or protocol for the equipment or reagent that you're using. Sometimes the manual for the equipment you're using provides the best introduction to a technique. When I was learning about peptide synthesis I didn't get the clearest explanation from a textbook or study, I got it from the manual for the peptide synthesizer in my lab. Most manufacturers have the PDF version of the manual online (FOR FREE! GRAB ALL OF THE FREE STUFF!). Pay special attention to the troubleshooting section because everything will go to hell eventually and at least you'll know why nothing worked.

3. Read studies effectively. Based on my experience you need to read a study at least twice to really understand it. There is a typical reading order: Abstract-->Introduction--> Results--> Discussion--> Methods and materials. When you read the results pay close attention to the captions for each figure because they will help you understand the data better. Passively reading a study is a great way to ensure that you remember absolutely nothing. It's a good idea to read and annotate the important studies. I use Mendeley to annotate and highlight my studies, but you can also use Acrobat Reader. 

Side note: The real reason I use Mendeley is because it lets me highlight everything in lots of pretty colors. I have and always will—love pretty colors. 

I have one comment summarising each section (e.g. Introduction, different parts of the results etc.) 

4. Sometimes you look at a method used in a study and it has an intimidatingly long and complex sounding name, you think, "The hell is THIS? Did they just throw a bunch of sciency words into a hat and use convoluted satanic rituals to get their results?" If you don't really understand or know about a technique find the original study where the researcher discovered the method. The OG study provides you with context and applications for that technique.

5. Are you daydreaming during a long train ride or waiting for a reagent to arrive or you're running an experiment that takes forever? Do your reading! When you're stuck waiting for results don't waste your time! Keep reading and reviewing literature.

6. Ask for help. Sometimes you try your best to understand a concept and it still doesn't sink in. Ask the Ph.D. students, post-docs and other equally clueless undergrads in your lab for help.

Those are my tips for doing all the reading and background research for a research project, but next time we will dive into writing and editing hell. 

The fate of your first draft.

[neurosciencestuff]

Seeing sounds: researchers uncover molecular clues for synaesthesia

1 in 25 people have synaesthesia, perceiving the world in unusual ways. An experience with one sense automatically leads to perception in another sense: for example, seeing colours when listening to music. Now researchers at the Max Planck Institute for Psycholinguistics and the University of Cambridge report clues into biological origins of such variations in human perception. They studied families with synaesthesia, and describe genetic changes that might contribute to their differences in sensory experience.

Some people with synaesthesia may see sounds, while others may taste them or feel them as shapes. This kind of sensory cross-talk comes in many forms, and develops during early childhood. It has been known for over a century that synaesthesia runs in families, giving a strong hint that inherited factors are important.

“Brain imaging of adults with synaesthesia suggests that their circuits are wired a little differently compared to people who don’t make these extra sensory associations. What we don’t know yet is how these differences develop,” said Dr. Amanda Tilot, a geneticist at the Max Planck Institute for Psycholinguistics. “We suspect some of the answers lie in people’s genetic makeup.”

A genetic window into sensory experience

In a report published in the journal Proceedings of the National Academy of Sciences, scientists from the Max Planck Institute for Psycholinguistics and the University of Cambridge report new genetic clues that could help explain the biology of synaesthesia. The researchers carefully analysed the DNA of three families in which multiple members, across several different generations, experience colour when listening to sounds.

The team took advantage of advances in genome sequencing, enabling them to identify genetic variants in the synaesthesia families and track how they were passed on from one generation to the next. In particular, they focused attention on rare DNA changes that altered the way genes code for proteins, and that perfectly matched the inheritance of synaesthesia in each of the three families.

While the highlighted DNA variants differed between the three families, a common theme emerged to connect them: an enrichment for genes involved in axonogenesis and cell migration. Axonogenesis is a key process enabling brain cells to wire up to their correct partners.

Combining families to uncover biological processes

Professor Simon Fisher, Director of the Max Planck Institute, who led the research, said, “We knew from earlier studies by the Cambridge team that no single gene can account for this intriguing trait; even families who experience the same form of synaesthesia are likely to differ in terms of specific genetic explanations. Our hope was that the DNA data might point to shared biological processes as candidates for involvement in synaesthesia.”

Professor Simon Baron-Cohen, Director of the Autism Research Centre, Cambridge University, commented, “This research is revealing how genetic variation can modify our sensory experiences, potentially via altered connectivity in the brain. Synaesthesia is a clear example of neurodiversity which we should respect and celebrate.”

In search of synaesthetes

To better understand these findings, the team is looking for new families and individuals to join their study. To learn more about their research and take a short test to find out if you experience a common form of synaesthesia, go to www.mpi.nl/synaesthesia.

[lifesciencepotluck]

I don’t know but I have a bizarre fascination with synaesthesia. 

[lifesciencepotluck]

Reverse Phase Chromatography

The ability to purify samples and isolate specific proteins is an important first step for studying proteins. If you want to get the 3D structure of a protein or an amino acid sequence or gain any understanding of how a protein interacts with something, then a basic requirement is having a pure sample. But let's face it your sample has a bunch of other undesirable junk, because nothing can be too convenient in science. Even if you chemically synthesize the protein instead of stealing it from the original source (like a dead anemone) you still have to purify the sample and get your target protein.

How do you do this?

There are a variety of liquid chromatography techniques used for purifying proteins such as anion exchange chromatography (separates proteins based on charge), affinity chromatography (this is really specific since the column will only bind to the antibody or ligand you choose, not just any randy that passes by), Size Exclusion Chromatography (separates proteins based on size) and Reverse-phase chromatography. These techniques exploit a specific property of the protein such as charge, size, isoelectric points etc. to separate proteins. Reverse-phase liquid chromatography (RP-HPLC) is one the most important and commonly used forms of liquid chromatography. It separates proteins based on how hydrophobic (water hating) or hydrophilic (water loving) a protein is. 

There are two phases in all types of chromatography. The stationary phase (or the lazy phase which clings to things) and the mobile phase, which slowly seeps through the stationary phase. For Reverse-phase the stationary phase is non-polar (or hydrophobic). The interaction between the stationary phase and analyte is mostly hydrophobic. So, if your protein is really hydrophobic (i.e. it has a lot of valine and alanine resides) then it will cling to stationary phase for its dear life until it is eluted by something more hydrophobic. Typically, the stationary phase in Reverse-phase columns have little beads made of silica gel and you have alkyl groups (hexyl, butyl, or ethyl groups) covalently linked to the beads. These little extensions are non-polar and they play a part in the hydrophobic interaction with your sample. The types of alkyl groups attached vary depending on the type of column you’re using and the kind of protein you're purifying. Of course, there are other types of beads like polymer based beads, which are better if you have the pH of your sample or solvent is above 7. BUT they are not as efficient at separating things.

Figure 1: Silica resin with C18 chains. 

Source: http://chem-net.blogspot.com/2013/11/reversed-phase-chromatography.html

The second player is the mobile phase or the hydrophobic solvent you will use to elute your protein. Usually as reverse phase happens you increase the hydrophobicity of the solvent and over time the proteins are eluted based on hydrophobic they are. The hydrophilic proteins come out first and the most hydrophobic proteins come out last because you need a higher concentration of your non-polar solvent. Most of the time, a mix of acetonitrile and trifluoroacetic acid (TFA) is used for the mobile phase. 

Acetonitrile (ACN) is a non-polar solvent. If you want to make the mobile phase more hydrophobic to elute the more hydrophobic proteins, then you increase the concentration of ACN. TFA is a weak hydrophobic ion-pairing reagent which helps maintain a low pH for our silica resin and helps reduce ionic interactions between the peptide/protein and the stationary phase (because there is always a small chance of something annoying like this happening). You can use other non-polar solvents like methanol, ethanol and isopropanol. Isopropanol works better for ridiculously large and hydrophobic proteins but it places a lot of pressure on the column. If the pressure is too high, your incredibly expensive column will die a horrible and resin-crushing death (Pro-tip: Always check the manual for the maximum pressure limit!).

You can tell how well your purification went based on the chromatogram. A chromatogram shows the UV absorbance for your sample against the retention time. Acetonitrile absorbs UV light very well which is why it's a great solvent for reverse phase. The peptide bonds in proteins absorb UV light at 215 nm whereas, aromatic amino acids like tryptophan absorb UV light at 280nm.  UV absorbance can be used to find the concertation of your protein or it can help determine the resolution for your feeble attempts at purification. 

If you have ‘peaks’ or ‘mini mountains’ that are well separated then your peptides are probably separated pretty well. But if your all of peaks on the chromatogram merge into an indistinguishable and horrifying blob—you have very poor resolution and the eluted sample you get at the end won't be that pure. You can take all of the eluted samples and try purifying them with even more reverse phase or other methods, but you will lose a bit of protein at each purification step. Or you can tweak your method to improve the resolution for reverse phase. 

Figure 2: What a chromatogram looks like in an ideal world where the experiment actually works. The “peaks” are sharp, narrow and distinct. 

Figure 3: A blob monster of my own making. I have probably spent more time doing Reverse phase than socialising at this point and I still get chromatograms like this. 

Stuff you need to worry about:

What kind of column your using: Sometimes you want to purify lots of your proteins in one go so you have to pick a column that can handle a high load. Other times you are analysing the purity of your sample and using a sensitive analytical column makes more sense.  If you want to isolate a really hydrophilic protein then using a c18 column is a solid option because it has more alkyl chains that can bind better to proteins that are not so hydrophobic. BUT since you are introducing a really strong hydrophobic interaction it can mess up the structure of your protein to the point where it loses its bioactivity. If you want to protect the bioactivity of your protein using a C4 or C5 column is a decent option.

Pore size: Each column will have its own pore size (the size of the gap between the beads) and you have pick the pore size based on the size of the target protein. Small pore sizes work for smaller proteins (<10kDa) but it most cases a pore size 300 Å works for a wide variety of protein sizes.

Temperature: A high temperature can speed up chromatography and give you better resolution (AKA the sharp and sexy peaks). All of this will come at the cost of your protein being denatured—of course.

pH: Proteins are pH sensitive since the amine, carboxylic and R groups of the amino acids can change charge depending on the pH.  Silica columns have a pH of 2-7, so the basic amino acids exist as positive ions. You need a hydrophobic anionic (negatively charged) ion-pairing reagent (like TFA) that will pair up with the positively charged basic amino acids, since you don't want any electrostatic interaction between the column and the peptide. (remember we only want to separate the proteins based on how hydrophobc they are)

Flow rate: A faster flow rate will allow a better separation. (Pro-Tip: Check the manual for the maximum flow rate. Usually the big, thick columns tend to have a higher flow rate than the thin, small and dainty columns)

Gradient: This is the most important factor which impacts the resolution. If you have a gradual increase in ACN concentration or hydrophobicity of your solvent (AKA a shallower gradient), you tend to get better resolution and higher peaks. However, if the run time is too long then your protein can get damaged since it’s being exposed to harsh chemicals (e.g. Acetonitrile) for a longer period of time.

There are other applications for Reverse-phase aside from protein purification. It isn't just used to purify and analyse proteins but it can work for drugs, metabolites, fatty acids, aldehydes and ketones. I hope this was a helpful introduction to Reverse-phase for anyone who is interested in the technique or actually has to use this technique. 

Next time, I’ll dive into Size Exclusion Chromatography (SEC).

Primary sources:

Dorsey, J. G. & Dill, K. A. The Molecular Mechanism of Retention in Reversed-Phase Liquid Chromatography. Chem. Rev. 89, 331–346 (1989).

Nagy, K. & Vékey, K. Separation methods. Med. Appl. Mass Spectrom. 61–92 (2008). doi:10.1016/B978-044451980-1.50007-0

Recommended readings:

Chromatography, R. Reverse-Phase Chromatography. Proteins 26–34 (2009). doi:10.1201/NOE1420084597.ch409

Bradshaw, T. P. A Users Guide: Introduction to Protein and Peptide HPLC. (2006).

[lifesciencepotluck]

Protein chemistry 101

Aside from obsessing over anemone toxins I have a soft spot for protein chemistry and protein biophysics in my heart. Luckily for me, I ended up in the protein chemistry lab at my university and I got to pick up some the techniques in protein chemistry. In my series of posts tagged #Proteinchemistry101 I hope to provide a basic introduction to some of the most common and useful techniques in protein chemistry like mass spectrometry, reverse phase, peptide synthesis etc. If you want to gain a more in depth understanding of some of these techniques then hop to the recommended readings section for the posts. If you are a student who actually has to do this stuff, it helps to get the manual for whatever equipment you're using (E.g. Jupiter C18 column) because those manuals also provide an introduction to these techniques plus troubleshooting tips.

I’m starting out with Reverse-phase Liquid Chromatography (RP-HPLC) and Size exclusion chromatography (SEC). If there any specific protein chemistry techniques you want me to cover, drop me an ask.