An NMR approach to scanning protein surface

GÓMEZ, G.E.; BERNAR, E.M. ; ARÁN, M. AND DELFINO, J.M

Sierra de la Ventana, Buenos Aires

Congreso; XLIII Reunión Anual de SAB; 2014

SAB

The solvent accessible surface area (SASA) is a key parameter to understand protein conformation and interactions. The photoreaction of diazirine (DZN) with the polypeptide chain serves to estimate the size and nature of SASA. DZN, similar to water in size and shape, generates methylene carbene, an intermediate species that reacts unselectively with its molecular cage. We used DZN for investigating protein folding and for mapping interfaces in protein complexes (1-3). Mass spectrometry techniques allowed us to derive a quantitative signal proportional to the extent of modification of the sample (4). Here, we study the feasibility to detect methylated products by multidimensional NMR, an approach that does not demand cleavage and is potentially rich in conformational information. We used E. coli thioredoxin (TRX) as a protein model. Expectedly, on the basis of the larger extent of SASA, the dominant modification phenomenon is the methylation of amino acid side chains, giving rise  predominantly to insertions into C-H bonds. A solvent  accessibility profile of the protein can be derived from 1H13C and 1H15N-HSQC spectra.