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My latest paper explored the effects of perchlorate salts on the structure, stability, and activity of my model enzyme α-chymotrypsin. These results essentially formed the benchmark for my understanding of how perchlorate salts affect α-chymotrypsin. This gave me a solid platform from which I can relate all future work back to as a reference point. So what did I find?
Perchlorate salts are bad for biochemistry, but not equally so. The three perchlorate salts examined were sodium, magnesium, and calcium perchlorate. I found that all perchlorate salts reduced the activity and the melting temperature of my enzyme in a concentration dependent manner. In fact the enzyme totally unfolds at room temperature in high enough concentrations of magnesium and calcium perchlorate. The extent to which these salts exerted a deleterious effect followed a fairly standard Hofmeister series, namely that sodium perchlorate exhibits the least deleterious effects, with calcium perchlorate exhibiting the most, and magnesium perchlorate exhibiting intermediate values. However, it is hard to draw solid comparisons between sodium perchlorate and magnesium and calcium perchlorate as they are divalent cations. Due to their stoichiometry, a one molar solution of magnesium or calcium perchlorate has twice as many perchlorate anions in solution as does a one molar solution of sodium perchlorate. You can of course double the amount of sodium perchlorate to equal out the anion concentrations, but now you have twice as many sodium cations in solution as you have magnesium or calcium. Ah the fun times we have trying to understand ionic effects in salt studies… This is why I prefer to look at these effects from a “whole salt” perspective.
Another feature explored in this study was the fact that the perchlorate effect appears to be dominant. On Mars, it is unlikely that an environment would contain only one type of salt, so it makes sense to examine combinations to assess whether they negate, or exacerbate each other. As magnesium sulphate is fairly widespread on Mars, it made sense to include it alongside the perchlorates. I found that magnesium sulphate increased the melting temperature of α-chymotrypsin, i.e. it stabilised the enzyme, but could it still exert this effect in the presence of perchlorates, or at least mitigate some of the perchlorate nastiness? Interestingly the answer was no, the perchlorate salt effect was dominant. This suggests that even if there are stabilising salts in a perchlorate containing solution, the deleterious effect of the perchlorates will still come out on top and exert its effect on biochemistry. This result was a little surprising as it has been shown previously that stabilising agents can generally negate the effects of a range of deleterious compounds. It is hard to explain why the perchlorate effect is dominant without understanding what is happening at the molecular level, which unfortunately is beyond us for now. If I were to speculate, I would suggest that the mechanism through which perchlorates exert these destabilising effects is not simply just the opposite of how stabilising molecules exert their effects.
This research builds on a long history of deleterious salt effects, otherwise known as chaotropicity. The deleterious nature of perchlorate salts agrees with many other studies, however the dominant nature of these effects was unexpected. Altogether this paints a rather bleak picture for the potential habitability of Mars if we cannot have functioning biochemistry in low molal concentrations of perchlorates. However, my research is all about the weird things that happen when we look at more than just one environmental parameter. We already saw that high pressures increased enzyme activity when in the presence of perchlorates, and I can only hint that we have found another interesting effect when you change a certain physical parameter. For now you will just have to believe me when I say that the possibility of biochemistry in perchlorate brines may not be as bleak as has been previously thought.
If the surface of Mars is such a hostile place, is the deep subsurface any better for life? This is the question that we have recently started to answer with our new publication in Communications Biology from myself, Michel Jaworek, Roland Winter and Charles Cockell.
So it’s fairly uncontroversial to say that the surface of Mars isn’t a nice place to be. The temperature fluctuates from ok to extremely cold, there’s constant incoming radiation, it’s salty, dusty and maybe most importantly there is essentially no liquid water. All in all, not the first place you would expect to find life on Mars. However, deep beneath the Martian surface there may be environments that are less hostile to life. Examples of such environments would be the subglacial lakes reported by Orosei et. al. in 2018 and Lauro et. al in 2020 or the deep ground water as suggested in Clifford et. al. 2010.
These deep environments are however not without their own challenges. Firstly, they would be extremely cold, and I mean cold. We are talking about environmental temperatures lower than 210 K (-63 °C), which is pretty far below the freezing point of water. But wait, lakes have been found beneath Mars, so how can they contain liquid water if it’s so cold? Well that leads us to our second problem, the salts. For liquid water to exist at such low temperatures we need salts in the solution to depress the freezing point of water low enough so that it doesn’t freeze. So it’s cold salty water, how bad can it be? Well the reigning champion of freezing point depression is our nasty friend the perchlorates, and for water to be liquid at such temperatures it has to be packed with them. Perchlorate salts aren’t something you come across much on Earth unless you’re from the Atacama or work at a munitions factory. On Mars however, they’re everywhere, and they’re bad news for life. We also have a third contestant in the extreme environmental parameter contest, and that is the high pressures which you would experience in the deep subsurface, and importantly, we just don’t know how life responds to both pressure and perchlorate salts.
So what did we do? Well, being biochemists we immediately ignored life itself and decided to look at a model enzyme and examined how its stability and activity changed with increasing concentrations of magnesium perchlorate and increasing pressures. Unsurprisingly we observed that magnesium perchlorate reduced the activity of our enzyme at ambient pressures, and we can use this as our starting point to compare our further observations to. So what happened when we increased the pressure? We found that the activity of our enzyme increased with pressure, even in the presence of perchlorate salts, and it was interesting to note that the greatest proportional increase happened with 0.25 M magnesium perchlorate. I’ll not go into the detail, but the reason why this happened is due to the fact that our enzyme has something called a “negative activation volume”. This essentially means that throughout the course of the enzymatic reaction, the volume of the enzyme decreases, and pressure loves it when you decrease the volume of something, and so it favours the enzyme reaction, thus increasing the activity. So if life on Mars was trying to avoid the hostility of the surface, it could gradually work its way into the subsurface, and if its vital biochemistry had a negative activation volume, it could expect to see an increase in its biochemical activity, effectively undoing the negative effects of the perchlorate salts.
It wasn’t a complete victory over the perchlorate salts though. We found that the salts reduced the temperature at which our enzyme melted and also lowered the pressure at which it unfolded. So while you can recover enzymatic activity that was lost due to the perchlorate salts, the folded phase space of our enzyme was ultimately constricted in the presence of perchlorates. This may have important implications. When you look at the phase diagram for life, it looks surprisingly similar to the phase diagram of proteins, as opposed to lipids or DNA. This means that protein stability is a good proxy for the feasibility of life, and our results suggest that perchlorate salts will constrict the possible environmental conditions in which life can survive. So it’s not all good news.
As with any study there are caveats. The concentrations of perchlorates that we used were far from the saturation point. This is simply because saturated magnesium perchlorate obliterates life and it will be a monumental amount of work (or a stroke of luck) to find life or biochemistry that can function at such high concentrations of perchlorates. Secondly, we had to ignore the low environmental temperatures and this was largely a technical consideration. Most lab equipment is designed to work really well at room temperature and ambient pressure. So making a spectrophotometer that works at 2 kbar is hard enough without also asking it to cool down to 200 K. However, watch this space, as the low temperature work is underway and will hopefully be coming to a publication near you soon….or however long peer review takes.
As most labs have been shut down due to COVID-19, it has been pretty hard to do research the past few months. Instead, I have been using this time for reading, writing, and familiarising myself with online bioinformatics software. Normally I get a bit scared when I hear about computational biology or chemistry, largely because the world of coding and modelling is still quite foreign to me. So it has come as a relief to find online bioinformatics software that is easy to use and interpret. I thought that it would be a good idea to run through some of the things I have been using recently, the databases they rely on and explain how to use them and interpret their results.
PDB
The Protein Data Bank (PDB) is a phenomenal database of over 160,000 protein crystal structures and all the bioinformatic software that I have used relies heavily on this great tool. The PDB is easily searchable and its greatest limitation is that you have to hope someone has determined the structure of your target protein. The PDB stores protein structure entries as 4 figure code, for example the SARS-CoV-2 spike protein is stored under 6VXX and this is its PDB ID. On the structure’s page you can then get lots of information about your target protein including, the year it was determined, the paper associated with it, its crystal score, its UniProt ID (more on this later) and perhaps coolest of all, the 3D view of your protein. The 3D viewer is great for understanding more about the actual structure of the protein you are interested in. You will be able to see the alpha helices, beta sheets, hydrogen bonds, bound cofactors etc. You can change the view from cartoon to molecular surface which is a great way to visualise active sites and deep binding pockets (which is especially nice if the structure comes bound with a ligand, you will see exactly how it fits in). The PDB is a great starting place for protein based bioinformatics projects.
UniProt
UniProt is the second bioinformatics tool on the list and like the PDB, it is absolutely massive. There are so many functions and uses for UniProt and I only know some, so hopefully I can do it justice. UniProt will provide you with a wealth of information about your protein of interest, provided the information exists. When I do enzymatic studies, I use bovine chymotrypsin, and you can see its UniProt entry here. On the page you can see that there is information such as its active site residues, where it acts in relation to the cell, post-translational modifications, multiple solved protein structures and the amino acid sequence. So obviously this is a quick and simple way to see what your enzyme does, what it acts on and where it does this. UniProt also has an align tool which essentially lines up proteins of your choice and tells you what residues are conserved, and which ones are biologically similar (pretty handy for approximating active sites). The align tool also draws a phylogenetic tree which is kind of cool, but I haven’t had a use for it yet. All in all it is great for getting more information about your protein of interest.
PoPMuSiC and HoTMuSiC
PoPMuSiC and HoTMuSiC are two pieces of protein stability prediction software which are incredibly useful beyond being brilliantly named. Both pieces of software are available as webservers at this link. PoPMuSiC is a protein stability predictor in terms of changes to the free energy of folding (ΔGF)upon point mutation, whereas HoTMuSiC predicts changes to protein melting temperature (ΔTm) upon point mutation. To use the software you have to enter the PDB ID for your target protein (as it’s a structure based predictor), you can then select to either get the results from a single, user defined mutation, or you can do a systematic run which provides you with the stability changes for each amino acid residue replaced by the 19 other possibilities. As predicting greater stability was the original aim for the software, it also produces a handy graphical output of the sum of the stabilising mutations at each respective residue. If you happen to know the melting temperature of your protein you can enter it into the HoTMuSiC software, which slightly increases the accuracy of the results. Some of the papers from the makers of the software can found here, here and here so that you can see some of the work you can do with the software.
SCooP
SCooP is another piece of protein stability software, made by the same makers of PoPMuSiC and HoTMuSiC. SCooP is different in that it will provide you with a predicted protein stability curve along with values for the upper melting temperature, the free energy of folding (at room temp), the heat capacity and the enthalpy change. What I really like about SCooP is that it is very visual, I really like being able to see something tangible and it is extremely intuitive. Here is a little demonstration of how it works which you can try for yourself using carbonic anhydrase as our example protein. Go to the site and enter these two PDB IDs, 4COQ and 5HPJ. These two proteins are homologous but come from different organisms. From the results see if you can tell which protein comes from an organism which lives in the cold depths of the ocean, and which one lives beside boiling hot water. You will see for yourself that SCooP is a great tool and the paper for it can be found here.
ProteinVolume
ProteinVolume is a bioinformatics tool which allows you to calculate the total volume, Van der Waals volume, void volume and packing density of your target protein. This is a great piece of software which is again incredibly easy to use. All you need to do is upload a PDB file to the server click go and wait for your results. The makers of the software have published some interesting pieces of work using the software which you can find here, here, and here.
This is just a short list of the bioinformatics tools and software that I have been using during the lockdown. There is plenty more out there, and websites such as OmicX and bio.tools are great resources for finding the right tool for your project.
I don’t think I’ve been totally converted to the ways of bioinformatics as I can’t wait to get back into the lab. I am however excited to keep up to date with bioinformatics as a field as I think the tools that come from it can really supplement my work and will hopefully provide directions for future experimental work and maybe even collaborations.
I often see posts online asking for advice on how to get into the field of astrobiology and have even had people reach out to me personally asking for advice. So I thought it might be useful to put all my advice into one place. Professor Lewis Dartnell also detailed his advice on his blog here. NASA’’s advice can be found here. To make it as applicable as possible I will cover things you can do when you are in school and then also at university. At this point it is important to note that my route to science has been very typical and focused and it is far from the only route into science. I will have posts in the future discussing atypical routes into science and astrobiology that my colleagues have taken. Most of this advice will be specifically relevant to science students, but hopefully non- scientists also find this useful. For now, here is my advice and my personal experiences with it.
#1 – Choose the Sciences and Keep Them for as Long as Possible
The easiest way to become a scientist is to study the sciences at school and for as long as possible. I know, fairly obvious. But I came very close to not studying biology for my GCSEs, the topic which would later become my career.
In third year I was picking my GCSEs and had provisionally picked physics and chemistry as my sciences. At the time I had no desire to keep on biology (I fully blame the curriculum for this situation) and had in fact chosen PE instead. It was then after a fateful PTA (Parent Teacher Association) meeting that my parents came home and told me that my biology teacher basically pleaded with them to have me pick biology as a GCSE subject. In the end I didn’t take much convincing and dropped PE in favour of biology. A decision, which in retrospect, set me up for the rest of my life so far. So thank you to my 3rd Year biology teacher, I owe it all to you. This forms the first part of my advice to you if you are at this early stage. Keep as many science subjects on as possible and for as long as possible. The doors these subjects open are vast and will give you plenty of options to pick for your A- levels and for university courses.
#2 – Know What Subjects you need for University
University is where you begin to become a specialist, therefore the courses have pre-requisites. Knowing what these are, is extremely important in picking your A- levels. Thankfully the sciences keep your options open and can branch into a large collection of degree courses.
For my A- levels, I broke from my own advice and picked biology, chemistry, history and politics. During my GCSEs I realised that physics and maths were just languages I couldn’t speak and so they had to go. This was somewhat of a departure from what a lot of my science/ medicine orientated peers picked, namely all three sciences and maths. My advice for picking your A- levels is to think about what you might apply to study at university and see what requirements those courses have.
#3 – Time for University
University is the gateway for most people into the world of science. Assuming you already know what subjects you are roughly interested in, your university degree should largely reflect those interests. People frequently ask what degree they should study to become an astrobiologist. I’ll explain later why it doesn’t really matter what you study specifically, just that you are interested in it. Being interested in your area will make all the work that much more bearable.
For university I applied to both UK and Irish institutions. I chose to attend Trinity College Dublin where I studied “science” for the first two years before specialising in biochemistry in my final two years. Here I was able to further develop my interests in science and learnt some of the key techniques that I would need for research in my field.
#4 – What to Study to Become an Astrobiologist?
The answer to this, is that it doesn’t really matter. Astrobiologists study everything from the formation of planets and stars to the potential for human exploration of space. You can take almost any topic and apply it to space. So the key to becoming an astrobiologist is to find how your specific field of interest can answer questions related to astrobiology. If you like geology then there is plenty of rocks in the solar system to keep you interested, if you are a biologist like me there are plenty of habitats to interrogate for their habitability or even if you are a lawyer there is the ever looming question as to the legality of the actions of nations in space. Becoming an astrobiologist is then about how you frame the questions you want to answer.
I studied biochemistry at university. The main topics of my lectures were subjects like human health and disease, signalling pathways, metabolism and genetics. Not exactly astrobiology was it? The important thing was to then take the skills and knowledge gained from a primarily biomedical focused degree and apply them to astrobiology. This is what will allow you to become an astrobiologist. Very few places formally teach or lecture on astrobiology, so the key to becoming an astrobiologist is to apply your own skills and questions to the field.
#5 – Try to get Experience in the Lab
It’s one thing to be interested in science, but it’s another to actually do it. The day to day life of a scientist is extremely variable across fields, disciplines and individual projects. If you have an idea of an area that you are interested in, try to get a summer placement with a lab in that field, or even just a few days. This will expose you to some of the realities of research. You will get to meet and talk with active scientists and hear their advice. You will also see that the scientific method is a rather messy affair. Experiments fail for unknown reasons, equipment breaks, orders are delayed amongst other unpredictable chaotic events. It’s rarely a smooth road in research. Having research experience also looks good on PhD applications.
I was lucky enough to secure a few summer research placements and can truly say they were a massive benefit. I was able to get hands on experience with techniques that I didn’t have access to, either at school or university. The placements also afforded me the opportunity to discover that I actually enjoyed doing research.
For examples of funding sources for summer research placements, see the Nuffield Research Placement, BBSRC Research Experience Placement and this list compiled by the BNA for examples. This is a non-exhaustive list and more sources of funding exist. Also note that labs may already have funding allocated for summer placements and they just need people to reach out to them to fill those spots.
#6 – Find Your Passion
One day you will be sitting in a lecture hall, probably half asleep, and the lecturer will say something that absolutely blows your mind. If the nugget of information that they just professed utterly consumes you for days on end and all you can do is think about it, then you have just found your passion in science. Finding something like this in your field of study gives you an avenue to formulate questions through.
For me, this happened when I got a copy of “On the Origin of Species” in school and was blown away. As I am sure you know, the school curriculums are rather focused on presenting facts which you can recite in an exam to the best of your ability. The topic of evolution was also taught terribly in Northern Ireland, as in, it was largely absent. So when I came across Darwin’s seminal work, I discovered my passion. I became obsessed with evolution and how, if there is an evolution there must be an origin, and if there was an origin here on Earth, could it have happened elsewhere. That final question is what lead me to the field of astrobiology.
On a quick side note, when you google UK astrobiology, the top result is the UK Centre for Astrobiology (UKCA) at the University of Edinburgh. I called this blog “The Irish Astrobiologist” because when you google Irish astrobiology, there is nothing, nada, zilch. Just links to the UKCA. I wanted to then have something to appear for my fellow students from Ireland, north and south, when they went to investigate this topic and to act as a focal point where people could reach out to ask me about astrobiology. Anyway, moving on.
#7 – What to do After Undergrad?
Once you have obtained your undergraduate degree, there are a multitude of things you can do. You can take time away from academia and studying in general. This time can be used to travel, work or even take some much needed time for yourself.
If you wanted to work but keep it orientated with your long-term career goals, a position such as a research assistant might interest you.
If you want to continue studying, then consider something like a master’s degree. They are becoming increasingly common in the UK and sometimes needed if you want to do a PhD in Europe. They can be a good opportunity to study something that is a little bit outside of your field to help you broaden and expand your knowledge and to get more time in the lab. They can also be useful to start generating ideas for future PhD projects and gaining skills and experience that can help you in the future.
Taking some time to figure out exactly what it is you want to do with your future can also be extremely beneficial in the long run. The decision over whether or not to do a PhD is one that should not be taken lightly. A PhD is a multi-year commitment and it may involve moving away from home to a new city or even country. In the end, you need to be sure that it is the right choice for you and that you are choosing to do one for the right reasons.
Ultimately there are many things you can do, and some things you may need to do due to personal circumstances. The main point I want to get across is that there isn’t any time pressure at this stage. You can take the time that you need before moving on to the next stage of becoming an astrobiologist. I will post interviews with colleagues who have taken breaks from university and academia to illustrate this point in the future.
#8 – Apply for a PhD
Obtaining a PhD is a goal for most scientists wanting to stay in academia. You will want to reach out to as many research groups as possible to enquire about PhD positions and the earlier the better, roughly a year before you plan to start. To find labs, look at papers which you found interesting and scan the author list to see which institutions the groups came from. The Astrobiology Society of Britain lists institutions which partake in UK based astrobiology research here, but it is unlikely to be an exhaustive list. NASA Astrobiology have their own list here, as does the European Astrobiology Network Association, EANA, here.
I reached out to many labs, some replied, some didn’t. I applied for five PhD programmes and was initially rejected from all of them, even the one I am in today. Thankfully another pot of funding appeared, and I was fortuitousness enough to then be accepted into the University of Edinburgh as a PhD student. Ask your lecturers and professors for help with applications, they have all been through this before and likely receive applications every year, so they know what they are talking about.
Your PhD project doesn’t have to be exactly what you did at undergrad, there is room to grow and develop your knowledge and skill. You aren’t meant to know everything going into your PhD and no one will expect you to.
#9 – Enjoy Yourself
The most important thing is to enjoy yourself. The road from school to university and beyond is a long one. You have to look after yourself. It can be easy to get obsessed with trying to achieve every metric of success or to compare yourself with the progress of others. This is bound to end in burnout. Mental health is rightfully becoming a prominent topic of discussion in academia. I consider myself lucky in that I have not had to brunt the worst that academia has to offer. I know amazing scientists and truly wonderful people who have been hurt by this system. The system is broken, so the most important thing you can do is look after yourself and those around you.
Hobbies, sports and other pursuits will help you stay grounded and give you an escape from the constant academic demands. For me, friends have been an invaluable source of joy and inspiration, without whom, I would never have completed my undergrad. We are all human and we are just doing the best we can.
In summary, if science is your thing, then study it as far and as wide as you can. Find something about astrobiology that you are passionately interested in and the research questions will come to you. It also does not matter what your background is, space is an astonishingly big place, and there is almost certainly an astrobiology question that can be answered by your area of interest. Enjoy the journey.
When asked what I research, the response “astrobiology” normally raises an eyebrow or two. It is then invariably followed by the question as to what exactly is astrobiology? Normally I’d wheel out the description employed by NASA that it’s the study of the origin, evolution and distribution of life in the universe and we would move on from there. However, I always feel like that description is one made from necessity for writing grants and other administrative literature. It fails to find what is truly exciting and interesting in what is an utterly wonderful field of research to be in. To give you my perspective, I will try and elaborate on why I find Astrobiology so interesting and what questions I seek to answer in my research. To achieve this, it is easiest to explain what I study and then hopefully convince you that it is interesting.
The question that I spend most of my time thinking about is, if life exists on Mars, what might it look like biochemically? As a biochemist, my interests are naturally biased towards such a question. What I find fascinating about Mars is that it harbours environments unlike any seen here on Earth. Therefore, it asks questions of biology which make no sense to ask if we were to solely focus on terrestrial life. Fortuitously for me, as it makes no sense to ask these questions, it means nobody has answered them, which produces a perfect little niche to fill with my PhD thesis.
So what environments am I talking about and what do they look like? The environments that I mainly focus on are concentrated perchlorate brines which are aqueous environments with high concentrations of perchlorate salts. Perchlorates salts are strong oxidising agents and are particularly lethal to life (unless you happen to be the subset of life that can eat small quantities of perchlorate). These environments have been hypothesised to exist deep underground in the Martian groundwater or even as a subglacial lake under the Martian south pole due o their extremely low freezing points (roughly -70 °C). So in addition to being rich in perchlorate salts, these environments have two other factors to keep in mind, namely the high pressures and the extremely low temperatures. This interplay between perchlorate concentration, pressure and temperature is something that we just fundamentally do not see here on Earth.
Recently, I have been studying how the perchlorate and pressure interplay affects a model enzyme. From this work (which will hopefully soon find its way past peer review) and the work of others, I have been able to develop a hypothesis about the biochemical nature of potential Martian life. This is the part of astrobiology that I find amazing. From the results of simple benchtop experiments, the application of biophysics and a sprinkling of evolutionary theory we are then able to derive scientifically valid conclusions about what might be happening deep underground on a rocky red planet millions of kilometres away. What is more amazing is that I can then go on and test this hypothesis and I thoroughly plan to do so. So whilst life on Mars may be totally novel in its biochemical ways (although I doubt it’s anything too novel), the fact that we are at a place in time where these questions can escape from the prison of thought experiment and have life breathed into them through experiment is utterly astounding to me. If and when the results get published, I can then go into them in greater length, so for now you’ll have to bear with me.
Therefore astrobiology is, to me, a scientific mindset from which we can explore questions about biology which we would never have asked, had we not looked out into the night sky. Along this line of enquiry we are bound to find weird and wonderful facets of biology and biochemistry not previously thought possible. It would be even more interesting though to find that the answers should not have been as unexpected all along.
