INVESTIGADORES
GOMEZ Gabriela Elena
congresos y reuniones científicas
Título:
STUDYING INTERFACES IN PROTEIN-PEPTIDE COMPLEXES AND PROTEIN CONFORMATION WITH A GENERAL PROBE COUPLED TO DETECTION BY MASS SPECTROMETRY
Autor/es:
MUNDO, M.R.; GĂ“MEZ, G.E; DELFINO, J.M
Lugar:
Buzios, Rio de Janeiro, Brasil.
Reunión:
Congreso; VII Iberoamerican Congress of Biophysics; 2009
Resumen:
Diazirine (DZN) is a photoreactive gas similar to water in size. Upon photolysis it generates methylene carbene (:CH2), which reacts unselectively with its molecular cage, inserting even into C-H bonds. Methylation depends primarily on the solvent accessible surface area (SASA) of the polypeptide chain. 3H-DZN was used in our laboratory for studying protein folding (1, 3) and for mapping interfaces in protein complexes (2). We investigated the feasibility of a non-radioactive methylene labeling procedure coupled to detection by 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. Here, we applied this technique for studying interfaces choosing as a model the complex calmodulin-melittin. We observed that the EM of melittin decreased when complexed with calmodulin, in correlation with the expected decrement in SASA due to complex formation. Additionally, we probed with DZN the increment in SASA experienced by calmodulin in the presence of Ca2+. This method -which relies on a novel application of MS to the detection of methylated products-, allows a straightforward measurement of SASA.