GOMEZ Gabriela Elena
congresos y reuniones científicas
General methylation of the solvent accessible surface to probe protein conformation. A new chemical modification method coupled to NMR detection
Rio de Janeiro
Congreso; V Latin American Protein Society Meeting / III French-Brazilian Symposium on Bioscience.; 2016
Institución organizadora:
Protein Society
The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding, conformational change and interaction. Nevertheless, this fundamental parameter is not readily addressable. The reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides (i) allows mimicry of water because of its size, and (ii) shows limited chemical selectivity because it generates extremely reactive methylene carbene (MC). DZN was useful to probe folding states and interfaces in complexes (Craig 2002 Protein Sci, Gómez 2006 Protein Sci, Ureta 2007 Biochemistry, Craig 2009 J Mol Biol). Moreover, the extent of methylation derived from ESI and MALDI-TOF spectra of reacted proteins provides a useful metric to evaluate SASA (Gómez 2012 J Am Soc Mass Spectrom, Gómez 2015 Anal Chem). Analytical detection of products by NMR is advantageous because it does not demand cleavage of the polypeptide, opening a rich view on structure. The extent of reaction of MC at various sites across the surface of E. coli thioredoxin (TRX) could be defined. The dominant modification phenomenon involves methylation of amino acid side-chains, as attested by the enrichment of the aliphatic region in 1H-NMR spectra. 1H-15N-HSQC spectra reveal the different impact of side-chain methylation on backbone amide environments. In the unfolded state (8M urea) TRX shows an enhanced and general methylation profile, consistent with a broad exposure to the aqueous solvent. 1H-13C-HSQC spectra of TRX reacted with 13C-DZN uncover new cross-peaks corresponding to water exposed methyl groups. Because of its mild reaction conditions and strong focus on side-chain modification, the DZN labeling approach adds value to other footprinting methods, such as H/D backbone exchange and hydroxyl radical reactions. Collectively, NMR spectral data of MC modified proteins offers a fertile source of information upon which a full map of solvent accessibility can be built.