Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Thursday Speaker Abstracts

Scanning Protein Surface with a Solvent Mimetic Probe: an NMR Approach Gabriela E. Gomez 1 , Evangelina M. Bernar 1 , Martin Aran 2 , Jose M. Delfino 1 . 1 Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina, 2 Fundacion Instituto Leloir, Buenos Aires, Argentina. Changes in the solvent accessible surface area (SASA) of proteins underlie protein folding, molecular recognition phenomena, and assembly of complexes. Nevertheless, this fundamental parameter becomes elusive for experimental scrutiny. Methylene carbene (MC) reacts with polypeptides allowing an estimate of the magnitude and nature of SASA. MC arises from the photocleavage of diazirine (DZN), a tiny heterocycle similar in size and shape to water, thus able to exert solvent mimicry. MC is an extremely reactive species that inserts readily into X-H bonds (X=C, O, S or N). Coupled to radioactivity or mass spectrometry detection, DZN labeling becomes useful to probe folding and interactions. By contrast, multidimensional NMR does not demand cleavage of the polypeptide, potentially opening a rich panorama on structure. General methylation was assessed in E. coli thioredoxin (TRX), where the insertion of MC at multiple sites across the surface was verified. 1H-NMR spectra of progressively reacted TRX show a significant enrichment in the aliphatic region, supporting the dominant insertion event into C-H bonds belonging to amino acid side-chains. Remarkably, buried residues such as Val 16 remain unmodified. Consistently, 1H,13C-HSQC spectra uncover new cross-peaks corresponding to water exposed methyl groups, while concurrently protein methylene signals disappear. On the other hand, 1H,15N-HSQC spectra reveal the impact of side-chain methylation on backbone amide environments. Discrete alterations occur at indole groups of partially exposed Trp 28 and 31. By contrast, backbone methylation itself is a rare occurrence. Because of its mild reaction conditions and strong focus on side-chain modification, DZN labeling adds its unique value to current footprinting methods, such as H/D exchange and hydroxyl radical reactions. Collectively, NMR analysis of MC modified proteins offers a fertile source of information upon which a full map of solvent accessibility can be built.

38