Exploring protein interfaces with a general photochemical reagent

GOMEZ, G., CAUERHFF, A., CRAIG, P., GOLDBAUM, F. A. AND DELFINO, J. M.

PROTEIN SCIENCE

Año: 2006 vol. 15 p. 744 - 752

0961-8368

Protein folding, natural conformational changes, or interaction between partners involved in recognitionphenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptidechain. This primary event can be monitored by the differential chemical reactivity of functional groupsalong the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generatesmethylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneousand indiscriminate modification of its immediate molecular cage. 3H-DZN was successfully used in ourlaboratory for studying protein structure and folding. Here we address for the first time the usefulness ofthis probe to examine the area of interaction in protein–protein complexes. For this purpose we chose thecomplex formed between hen eggwhite lysozyme(HEWL) and themonoclonal antibody IgG1D1.3. :CH2labeling of freeHEWLor complexed with IgG1 D1.3 yields 2.76 and 2.32mmolCH2 per mole protein at 1mMDZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrementin the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), inagreement with the known unspecific surface labeling reaction of :CH2. Further comparative analysis atthe level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably,those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21,G117TDVQAWIR125, andG22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as afeasible methodology useful for mapping contact regions of protein domains involved in macromolecularassemblies.