IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
artículos
Título:
Exploring protein interfaces with a general photochemical reagent
Autor/es:
GÓMEZ, GABRIELA E.; CAUERFF, ANA; CRAIG, PATRICIO O.; GOLDBAUM, FERNANDO A.; DELFINO, JOSÉ M.
Revista:
PROTEIN SCIENCE
Editorial:
Cold Spring Harbor Laboratory Press
Referencias:
Lugar: Cold Spring Harbor, NY , USA; Año: 2006 vol. 15 p. 744 - 752
ISSN:
0961-8368
Resumen:
Protein folding, natural conformational changes or interaction between partners
involved in recognition phenomena bring 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) by irradiation at ë>300 nm. The extreme chemical reactivity of this
species allows the almost instantaneous and indiscriminate modification of its
immediate molecular cage, inserting even into C-H bonds. 3H-DZN was successfully
used in our laboratory for studying protein structure and folding (Craig P.O., Ureta
D.B., Delfino J.M., 2002, Protein Sci 11: 1353-1366). 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 that 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 mol 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 and G22YSLGNWVCAAK33. Thus, protein footprinting with DZN
emerges as a feasible methodology useful for mapping contact regions of protein
domains involved in macromolecular assemblies.