Studying interfaces in protein-peptide complexes and protein conformation with a general probe coupled to detection by mass spectrometry. ARTICULO ENVIADO PARA SU PUBLICACION

GÓMEZ, G.E (BOTH AUTHORS CONTRIBUTED EQUALLY TO THIS WORK); MONTI, J.L (BOTH AUTHORS CONTRIBUTED EQUALLY TO THIS WORK); DELFINO J.M

JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY

ELSEVIER SCIENCE INC

Año: 2011

1044-0305

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 uponphotolysis generates :CH2, reacting unselectively with its molecular cage, inserting eveninto C-H bonds, and giving rise to stable methylated products. By virtue of thesefeatures, DZN acts as a reasonable molecular mimic of the aqueous solvent andmethylation depends primarily on the solvent accessible surface area (SASA) of thepolypeptide chain. 3H-DZN was used in our laboratory for studying protein folding andfor mapping interfaces in protein complexes. More recently, we investigated thefeasibility of a non-radioactive methylene labeling procedure coupled to detection byelectrospray mass spectrometry (ES-MS). The resolution of ES-MS spectra allowed usto distinguish M+n*14 peaks and to assess, in a quantitative fashion, the extent ofmethylation (EM) of the polypeptide chain. This parameter demonstrated to be sensitiveto the conformational state of proteins. Here, we applied this technique for studyinginterfaces and conformational features in proteins. For the first aim, we chose as amodel the complex formed between calmodulin and melittin. We proved that the EM ofcomplexed calmodulin decrease, in correlation with the expected decrement in SASAdue to complex formation. To assess conformational changes, we mapped out along theamino acid sequence the differential solvent exposure experienced by calmodulin in thepresence or in the absence of Ca2+. This method -which relies on a novel application ofES-MS to the detection of methylated products-, allows a straightforward approach tothe measurement of SASA.