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.