A view on conformation by general methylation of surfaces. An NMR approach.

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

CABA

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

A VIEW ON PROTEIN CONFORMATION BY GENERAL METHYLATION OF SURFACES. AN NMR APPROACH.Abstract: The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding and interactions. However, this fundamental parameter eludes direct analysis. Our approach involves the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. Because of its size, DZN (i) mimics water, and (ii) shows narrow chemical selectivity, due to the extreme reactivity of methylene carbene (MC). Detection of products by NMR benefits from the fact that it does not demand cleavage of the polypeptide. The extent of MC reaction at various sites across the surface of E. coli thioredoxin (TRX) was assessed. The preservation of conformation and stability of the labeled samples was evaluated by circular dichroism (CD) and fluorescence emission. The dominant phenomenon involves methylation of amino acid side-chains, as attested by the enrichment of the aliphatic region in 1H-NMR spectra. In the unfolded state, an enhanced and broad methylation profile points to extensive solvent exposure. 1H-13C-HSQC spectra of native TRX reacted with 13C-DZN reveal new cross-peaks corresponding to water-exposed methyl groups. 1H-15N-HSQC spectra reveal the different impact of the reaction on backbone amide environments. The relative intensity of CHα spots is indicative of the extent of methylation at individual amino acid residues. Moreover, CH, CHγ and CHδ cross-peaks pinpoint details of side-chain modification. A fully consistent pattern emerges from both HN and HC profiles. Outer as well as inner (cavity) surface components become prime targets of methylation. Because of its mild reaction conditions and strong emphasis on side-chain modification, the MC labeling approach emerges as a distinctive footprinting method. All in all, the newly gathered spectral data highlights a rich panorama on protein conformation. Keywords: protein conformation, solvent accessible surface area, footprinting technique, NMR, thioredoxin.