Surface methylation profiles unravel protein conformation. An NMR approach.

GOMEZ, GABRIELA E; DELFINO, J.M; BERNAR, EVANGELINA M; ARÁN, M; SMAL, C

Edimburgo, Escocia

Congreso; 19Th IUPAB congress and 1th EBSA congress; 2017

IUPAB-EBSA

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 mimics water because of its size, and 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 along 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. 1H,13C-HSQC spectra of TRX reacted with 13C-DZN reveal new crosspeaks corresponding to water-exposed methyl groups. 1H,15N-HSQC spectra show 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crosspeaks 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.