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Exploring protein interfaces with a general photochemical reagent

¹ Departamento de Química Biológica–IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina
² Fundación Instituto Leloir, C1405BWE Buenos Aires, Argentina

(RECEIVED November 10, 2005; FINAL REVISION January 11, 2006; ACCEPTED January 13, 2006)

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH₂). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. ³H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein–protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG₁ D1.3. :CH₂ labeling of free HEWL or complexed with IgG₁ D1.3 yields 2.76 and 2.32 mmol CH₂ per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH₂. Further comparative analysis at the 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 a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

Keywords: diazirine; methylene carbene; protein interfaces; protein complexes; solvent-accessible surface area; protein footprinting; epitope mapping; hen egg white lysozyme

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