Probing solvent accessible surface area in proteins: an NMR approach.

G.E. GÓMEZ; BERNAR, EM; ARAN, M; SMAL, C; DELFINO J.M

Salto Grande

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

SAB - Sociedad Argentina de Biofísica; Seccional Biofísica de la Sociedad Uruguaya de Biociencias

Changes in the solvent accessible surface area (SASA) of proteins underlie protein folding, molecular recognition and assembly of complexes. Nevertheless, this fundamental biophysical parameter is elusive in experimental measurement. Our approach to this problem relies on the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. This reagent (i) exerts solvent mimicry because its size is comparable to water and (ii) shows scant chemical selectivity because it generates extremely reactive methylene carbene (MC) that inserts readily into X-H bonds (X=C, O, S or N). Quantitative metrics resulting from ESI and MALDI-TOF spectra of reacted proteins and fragments derived thereof, allowed us to estimate the extent of methylation of samples. These measurements provide a unique way to evaluate the extent and nature of SASA, from which insights can be drawn into conformation and interactions1,2. Analytical detection of products by NMR is advantageous because it does not demand cleavage of the polypeptide, potentially opening a rich panorama on structural information. The general methylation process was assessed in E. coli thioredoxin (TRX), where the extent of reaction of MC at various sites across the surface could be defined. 1H-NMR spectra of native TRX samples progressively reacted with DZN show a significant enrichment in the aliphatic region supporting the expectation that the dominant modification phenomenon takes place at amino acid side-chains. Consistently, 1H,13C-HSQC spectra uncover the appearance of new cross-peaks corresponding to water exposed methyl groups, whereas concurrent methylene signals disappear. 1H,15N-HSQC spectra reveal the different impact of side-chain methylation on backbone amide environments. In addition, modification at indole (W) and amide (N, Q) side chains occur. By contrast, samples of TRX labeled with DZN in the unfolded state (8M urea) show an enhanced extent of methylation, consistent with a general exposure of the polypeptide to the aqueous solvent. Collectively, NMR spectral data of MC modified protein samples offers a fertile source of information upon which a full map of solvent accessibility of the protein can be built.