A Photochemical Approach To The Study Of Interfaces In Protein-Protein Complexes.

GÓMEZ G.E; CAUERHFF A; CRAIG P.O; GOLDBAUM F.A; DELFINO J.M

Florence, Italy

Simposio; Fifth European Symposium of the Protein Society; 2003

A photochemical approach to the study of interfaces in protein-protein complexes. Gabriela E. Gómez1, Ana A. Cauerhff2, Patricio O. Craig1, Fernando A. Goldbaum2, and José M. Delfino1. 1Departament of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, (1113), Buenos Aires, Argentina. E-mail: rtdelfin@criba.edu.ar. 2Leloir Institute, Buenos Aires, Argentina.   Changes in solvent accessible surface area occurring upon protein folding or upon interaction between protein partners involved in recognition phenomena can be monitored by the differential chemical reactivity of modifiable functional groups along the protein sequence. 3H-diazirine (3H-DZN), a photoreactive gas similar in size to water had been successfully used in our laboratory for studying protein structure and folding (Craig et al. 2002. Protein Science 11:1353-1366). We used this reagent to address the examination of areas of interaction between HEL (hen egg white lysozyme) and the monoclonal antibody IgG1D1.3. By irradiation at l>300nm 3H-DZN generates methylene carbene ([3H]:CH2), which reacts almost instantaneously and indiscriminately with its immediate molecular cage, inserting even into C-H bonds. HEL was labeled free or complexed with IgG1D1.3 yielding 0.0027 and 0.0022 mol CH2/ mol protein at 1mM DZN respectively. This amounts to 18% less in the latter case. Assuming a pure non specific reaction with the polypeptide chain, one might expect a correlation with the observed decrement in solvent accessible surface area due to complex formation (11%: Amit et al. 1986. Science 233: 747-753). Tryptic digests derived from modified HEL -free or complexed with the antibody- were separated by size exclusion chromatography and RP-HPLC. Peptides eluting in the molecular weight region where those implicated in the interaction with D1.3 should appear showed less label (8-25%). Thus, ¢protein footprinting¢ with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.