Surface methylation profiles unravel protein conformation. An NMR approach.

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

Edinburgh

Congreso; 19th International Union of Pure and Applied Biophysics (IUPAB) and 11th European Biophysical Societies' Association (EBSA) Congress.; 2017

IUPAB-EBSA

Surface methylation profiles unravel protein conformation. An NMR approach.EM Bernar*1, GE Gómez*1, C Smal2, M Arán2 and JM Delfino11Departamento de Química Biológica e IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires, Argentina 2Fundación Instituto Leloir, Av Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina*both authors contributed equally to this workThe solvent accessible surface area of the polypeptide chain plays a pivotal role in protein folding and interactions. However, this fundamental parameter eludes direct scrutiny. The reaction of the minute photochemical reagent diazirine (DZN) with polypeptides (i) mimics water because of its size, and (ii) shows limited chemical selectivity due to the extreme reactivity of methylene carbene (MC). Detection of products by NMR is advantageous because 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 dominant modification 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 general methylation profile points to broad 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, CH, CHcross-peaks pinpoint details on side-chain methylation. A fully consistent pattern emerges from both HN and HC regions. Collectively, the protein methylation profile provides a unique footprint on protein conformation.