One of the main issues with obtaining protein structures, especially that of transmembrane proteins, are their relative flexibility. This issue limits both of the most commonly used methods of obtaining larger protein structures: X-ray crystallography and cryo-electron microscopy (cryo-EM). Indeed, in their native environment, proteins are highly dynamic and cycle through different conformations depending on their immediate surroundings. However, X-ray crystallography can only obtain one protein conformation at a time and even the number of conformation obtained by cryo-EM is limited. Intermediary structures between the active and inactive form of a protein are particularly difficult to sample, resulting in a considerable bottleneck in the analysis of conformational changes.
Luckily, one particular spectroscopy method, pioneered 25 years ago by Altenbach et al. and which requires the insertion of paramagnetic probes, can help feel in the gaps. This Electron Paramagnetic Resonance (EPR) utilizes the ability of unpaired electrons to change between their two spin states (ms = ½ or ms = - ½) and the influence that the nearby environment and associated atoms has on them to derive a spectral line. One particularly useful application of this method for protein structural analysis is the double electron-electron resonance (DEER) measurements: the distance between a pair of labelled spins is measured, and then that distance can be measured again for the same protein under different conditions (for example in the presence of various ligands).
Nowadays, the most common way to establish spin labeling is by inserting reactive cysteines via site-selective mutagenesis at dynamically relevant sites in the protein of interest. The unpaired electrons within the accessible cysteines will then bind to the spin labels when incubated with the purified proteins under specific conditions.
My own project at the Scheerer laboratory will include performing this experiment on the melanocortin-4 receptor (MC4R) in different environments, such as in the presence of orthosteric and allosteric ligands, in collaboration with Matthias Elgeti’s laboratory at Leipzig University. So far, I am pleased to report that the wildtype MC4R has been successfully labeled by both MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) and IDSL (bis(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-il)-disulﬁde spin label). MTSL is a nitroxide reagent with a high reactivity towards cysteines. Its high labeling efficiency combined with its small size, which minimizes the risk of interference with the protein structure and the folding pathway, have made it the most popular spin label for proteins. The reactive disulﬁde IDSL, on the other hand, labels cysteines via sulfhydryl-disulﬁde exchange reaction. Additionally, an intra-side chain S-N stabilizes the interaction, making IDSL an attractive label for DEER measurements.
The next step is now to obtain a “cysteine-less” variant, which cannot be labeled for EPR, as a negative control. This represents one of the unfortunate drawbacks of EPR spectroscopy, as this process is time-consuming, and we have to account for the unpredictable possibility that this might result in a loss of expression or structure. This is what I am currently doing, generating two virus containing “cysteine-less” mutants of MC4R: one of them had four reactive cysteines mutated into serines and the other had five (the same four and one additional cysteine that is believed to undergo post-translational modification in humans). My hope is that at least one of these MC4R mutants will not only be expressed in a large enough amount to be used in subsequent experiments, but also that EPR will show no labelling of the remaining cysteines. Indeed, we consider that the remaining native cysteines, being either involved in a disulfide bond or hidden within the transmembrane core, should not be available for labeling. If this experiment is successful, I will then be able to move on to constructs with cysteines inserted at interesting sites, such as in the transmembrane regions 5, 6 and 7 and on the intracellular loop 2, and thus begin the DEER measurements in earnest.
Time goes by so fast, that’s undeniable. I remember the day I started my PhD as if it was yesterday. Here I am today, almost in the second year of PhD. At this pace, I have no doubt that in the blink of an eye, I will be writing my thesis.
When I look back, what I can say is this last year was extremely unique and memorable with its highs and lows. Well, you more or less know about my journey from my previous blogpost, but what I hadn’t told you, also what I didn’t know, at that moment was the challenges that a computational chemist has to go through topped with the mysterious world of experiments, not only in academia but also in the corporate world. Now please bear with me and let me tell you the tale of the “shapeshifter” (that’s what I like to call myself lately)
As someone who chose the computational chemistry path back in the sophomore year of college, I was slowly straying away from the idea of performing experiments in the lab again. My practical lab courses were long gone. As the time passed by, my skills regarding experimental methods got rusty more and more.
After I had an interview to get the ESR4 position, Prof. Carles Curutchet asked me whether or not I would also be willing to do some experiments for the project. I thought it would be great to do both the experimental and the computational part of the job to be able to own it completely. Though I cannot deny that I was a little scared. As I was gaining more experience and deepening my knowledge in theoretical methods, my ability in performing experiments in the lab was decaying at the same pace. I was afraid of getting lost in things which could be pretty obvious to some wet chemists but clearly not to me, terrified by even the thought of failing…
As human beings, when it comes to unknown things, we are always a bit anxious and scared, aren’t we? It is because we don’t exactly know what we are facing and how to deal with it, but it is also somewhat intriguing and exciting, don’t you agree?
So, long story short, I didn’t chicken out and said yes to performing experiments besides the computational work I was supposed to do. In a few months, I found myself re-learning the fundamentals of working in a lab (general chemistry lab 101 reloading…) I was cautious as if I could possibly press a button and burn the whole lab down. I asked for guidance, talked to the PhD students, professors, read the manuals of the machines I was going to use before taking action. For each type of experiment, I was supposed to use a specific machine which was located in different labs of the department. So, in the morning I was in one lab, in the afternoon in another one. Even people seeing me here and there were not entirely sure which lab group I belonged to or who I was working with. After some time, in the experimental section of the department, I became someone everyone was used to seeing around but nobody knew much about, and little did they know that I was actually a member of the computational chemistry group. Having two different roles also made it complicated to explain what exactly I was doing. Yet, it was just the tip of the iceberg.
In February, I started my secondment at Gain Therapeutics. The company was located only 5 mins away from the pharmacy campus of University of Barcelona where I normally work at. So, in a way it was different than the usual secondment concept where you move to another country for a couple of months and work at another institution while a new culture is being introduced to your system. For me it was regular PhD work + secondment in the same city all blended in together. There were days I stopped by the university in the morning to get a sample and then went to work. It just added one more location to the whole equation of where I work.
In the company, I got a nice desk in a nice office, like any other member of the computational team, but as a part of my secondment plan, I was also supposed to perform some experiments in the Gain lab which was completely on the opposite side of the office. After going down with the elevator, taking a long walk down the hallway, passing through two turnstiles, scanning your card twice, opening several doors, congratulations you made it to the lab (In this case is it short for laboratory or labyrinth). After a short while, I figured out that the machine they had in the Gain lab was not suitable for my experiments. So, I had to use the one in the common area which was one story below the Gain lab and required going through another door with scanning your card, though that door required another type of authorization which I didn’t have as a visiting researcher. So, every time I had to use a machine, I either needed someone to accompany me or borrow someone else’s card. While working there in the lab, I got into the world of biologists, because most people working in the lab had a biology background. Actually, they were surprised when I told them I was a chemist (just a chemist, let alone the computational part).
Here I am now, performing some experiments + simulations at academia & company.
I can be a computational or an experimental chemist or both, I can be at the university or appear at the company or both. It feels like I have some magical powers and I can shapeshift from one to another like a druid (gamer detected). So, at this point I am not sure how to explain where I work, what I do exactly, which team I belong to. When I get asked such questions, oh I can guarantee that there is a long story on its way, but to keep it shorter and make it sound more like me I will summarize my current PhD life with an acrostic:
Little did I know that I was about to start one hell of a journey
I thought I’d just be on my PC without getting my hands dirty
Fast-forward a few months, I started to conduct an experiment
Eventually I found myself at Gain exploring company environment
One day you can find me around the spectrometer in the lab
For the rest, I am doing my simulations at the office looking fab
Partially a student at school, partially a researcher at the company
Half wet chemist, half computational, not wearing just one hat, but so many
Don’t underestimate the life of a comperimental chemist in academpany