Conformation and interactions through the analysis of protein surfaces

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

CABA

Conferencia; Simposio Fronteras en Biociencia 2; 2016

Instituto de Biomedicina de Buenos Aires (IBioBA) - CONICET - Max Planck Society Partner (MPSP)

The 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. Collectively, the protein methylation profile provides a unique footprint on protein conformation.