Exploring protein interfaces with a general photochemical reagent.
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 (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-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 IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) 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 :CH(2). 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: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
Gómez
Gabriela E
GE
Departamento de Química Biológica-IQUIFIB, UBA-CONICET, Facultad de Faramcia y Bioquímica, Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina.
Cauerhff
Ana
A
Craig
Patricio O
PO
Goldbaum
Fernando A
FA
Delfino
José M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D003978:Diazomethane / Q000737:chemistry* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D015335:Molecular Probes; D010455:Peptides; D019920:Photoaffinity Labels; D011506:Proteins; C504223:methylene radical; D003978:Diazomethane; C421864:hen egg lysozyme; D009113:Muramidase; D008697:Methane