Mapping solvent accessibility in proteins. Insights into folding and interaction issues
The study of the topography of proteins and the interactions where they are involved can be addressed through photochemical mimicry of the aqueous solvent. Modification of the polypeptide chain provides insights on the folding state, because this measurement is a function of the size and nature of the solvent accessible surface area (SASA). Similarly, occlusion of SASA as a consequence of protein interactions allows one to map the sites involved. Methodology developed in our laboratory is based on the unspecific photoreaction of diazirine (DZN, the smallest CNN heterocycle) with proteins, coupled to the suitable detection of the addition (methylated) products. It is possible to estimate quantitatively the extent of modification by the use of radiotracers (tritiated DZN), or alternatively, by metrics derived from modern mass spectrometry techniques (MALDI-TOF and ESI-MS) or multidimensional NMR. With MS detection, maximal resolution of the labeled site is achieved after fragmentation of the polypeptide chain at the level of small peptides, or even individual amino acids. Interestingly, the NMR approach does not demand chemical or enzymatic cleavage and is potentially rich in conformational information. Predictably, on the basis of the larger extent of SASA, the phenomenon ruling the DZN modification is the methylation of amino acid side chains, giving rise predominantly to insertions into CH bonds. In this fashion, the probability of reaction at individual sites along the polypeptide reveals maps of the aqueous solvent accessibility. Through the analysis of the extent and quality of the photolabeled products, conformations can be distinguished corresponding to the native or intermediate states, or the average picture of the unfolded ensemble. On the other hand, a well-known paradigm of a peptide-protein complex (calmodulin-melittin) illustrates the value of DZN photolabeling as a differential foot-printing technique able to pinpoint the area of interaction, by the SASA occlusion occurring as a consequence of complex formation. One can hardly overemphasize the worth of these new methods for the benefit of structural proteomics and interactomics.