INVESTIGADORES
GOMEZ Gabriela Elena
congresos y reuniones científicas
Título:
Studying interfaces in protein-protein complexes with a photochemical probe.
Autor/es:
GÓMEZ G.E; CAUERHFF A; CRAIG P.O; GOLDBAUM F.A; DELFINO J.M
Lugar:
San Carlos de Bariloche, Río Negro, Argentina
Reunión:
Congreso; Bariloche Protein Symposium, XXXIX Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB), XXXII Reunión Anual de la Sociedad Argentina de Biofísica (SAB).; 2003
Resumen:
studying interfaces in protein-protein complexes with A photochemical probe Gómez G.E.1, Cauerhff A.A.2, Craig P.O.1, Goldbaum F.A.2, Delfino J.M.1 IQUIFIB (Universidad de Buenos Aires-CONICET). 2Instituto Leloir. E-mail: ggomez@qb.ffyb.uba.ar The importance of interactions between proteins in biology has made the molecular recognition process an area of considerable interest. A consequence of complex formation is a reduction in the solvent accessible surface area (SASA). Our approach is based on a general photochemical modification of the polypeptide chain, useful for addressing changes in SASA occurring upon protein interaction. 3H-diazirine (3H-DZN) is a photoreactive gas similar in size to water. By irradiation at l>300nm 3H-DZN generates methylene carbene ([3H]:CH2), which reacts with its immediate molecular cage, inserting even into C-H bonds. As a model system we used this reagent to probe the complex formed by HEL (hen egg white lysozyme) and the monoclonal antibody IgG1D1.3. HEL was labeled free or complexed with IgG1D1.3 yielding 2.76 and 2.32 mmol CH2/ mol protein at 1mM DZN, respectively. This agrees with the observed decrement in SASA occurring because of complex formation. Tryptic digests derived from labeled HEL -free or complexed - were separated by size exclusion chromatography and RP-HPLC. The measurement of radioactivity and the identification of each isolated peptide showed that those implicated in the area of interaction had the highest differential labeling (H15GLDNYR21; G117TDVQAWIR125; G22YSLGNWVCAAK33). Thus, ¢protein footprinting¢ with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.