Probing interfaces and protein conformation with a solvent mimetic photoreagent coupled to detection by mass spectrometry.

MUNDO, M.R.; GÓMEZ, G.E; DELFINO, J.M

Salta, Argentina.

Congreso; 3rd Latin American Protein Society Meeting and XXXIX Annual Meeting of the Argentinean Biophysical Society (SAB); 2010

Methylene carbene (:CH2) is one of, if not the, most reactive organic species known. Diazirine (DZN) is a photoreactive gas similar to water in size and shape, which upon photolysis generates:CH2, reacting unselectively with its molecular cage, inserting even into C-H bonds, and giving rise to stable methylated products. By virtue of these features, DZN acts as a reasonable molecular mimic of the aqueous solvent and methylation depends primarily on the solvent accessible surface area (SASA) of the polypeptide chain (1). 3H-DZN was used in our laboratory for studying protein folding and for mapping interfaces in protein complexes (2, 3). More recently, we investigated the feasibility of a non-radioactive methylene labeling procedure coupled to detection by electrospray mass spectrometry (ES-MS). The resolution of ES-MS spectra allowed us to distinguish M+n*14 peaks and to assess, in a quantitative fashion, the extent of methylation (EM) of the polypeptide chain. This parameter demonstrated to be sensitive to the conformational state of proteins (3). Here, we applied this technique for studying interfaces and conformational features in proteins. For the first aim, we chose as a model the complex formed between calmodulin and melittin. We proved that the EM of complexed calmodulin decrease, in correlation with the expected decrement in SASA due to complex formation. To assess conformational changes, we mapped out along the amino acid sequence the differential solvent exposure experienced by calmodulin in the presence or in the absence of Ca2+. This method -which relies on a novel application of ES-MS to the detection of methylated products-, allows a straightforward approach to the measurement of SASA.