Scanning protein surface with a solvent mimetic probe: an NMR approach.

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

Madrid

Congreso; Biophysical Society Thematic Meeting: ?Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling.?; 2015

Universidad Complutense de Madrid

process of protein folding and key biological phenomena such as molecular recognition between ligands and receptors and assembly of oligomeric complexes. Despite its central role in protein biophysics, this fundamental parameter becomes elusive from experimental measurement.The unselective reaction of methylene carbene (MC) with the polypeptide chain allows estimating the magnitude and nature of SASA. MC arises from the photocleavage of diazirine (DZN), a tiny and stable heterocycle similar in size and shape to water, thus able to exert solvent mimicry. DZN is a gas readily soluble in water, whose concentration can be accurately estimated by its absorption at 320 nm. Upon irradiation at λ > 300 nm -a UV region beyond that where natural protein cromophores absorb- DZN gives rise to MC, an extremely reactive electron deficient species that gives rise to insertion events into X-H bonds (X being C, O, S or N atoms), therefore showing scant chemical selectivity. DZN served the purpose of probing folding states and interfaces in complexes (Craig 2002 Protein Sci, Gómez 2006 Protein Sci, Ureta 2007 Biochemistry, Craig 2009 J Mol Biol). ESI and MALDI-TOF analyses of reacted products at the level of the full length protein, and after fragmentation into peptides or even at the level of individual amino acid residues allowed us to develop quantitative metrics useful for estimating the extent of methylation of samples (Gómez 2012 J Am Soc Mass Spectrom, Gómez 2015 submitted). These measurements provide a unique way to evaluate the extent of solvent exposure, from which insights can be drawn into conformation and interactions. By contrast with mass spectrometry, multidimensional NMR methods do not demand cleavage of the polypeptide, potentially opening a rich panorama on structural information. By using E. coli thioredoxin (TRX) as the target protein, the general unspecific methylation process was assessed. In this way, the extent of reaction of MC at various sites across the protein surface could be defined. Mono dimensional 1H-NMR spectra of TRX samples progressively reacted with DZN show a significant enrichment in the aliphatic region. This observation supports the expectation that -on the basis of the larger extent of solvent exposure- the dominant modification phenomenon is the methylation of amino acid side chains, predominantly giving rise to insertions into C-H bonds. Remarkably, buried residues such as Val 16 remain unmodified. Consistently, 1H,13C-HSQC spectra uncover the appearance of new cross-peaks corresponding to water exposed methyl groups, whereas concurrent methylene signals disappear. On the other hand, 1H,15N-HSQC spectra reveal the differential evolution of the backbone amide environments arising from the methylation of side-chains. Discrete alterations occur at the indole groups of partially exposed Trp 28 and 31. By contrast, methylation of the backbone itself is a rare occurrence. A case in point is Gly 65, that remains unaltered. Because of its mild modification conditions and its strong focus on side-chain modification, the DZN labeling approach adds its unique value to other current footprinting methods, such as those based on H/D backbone exchange and hydroxyl radical reactions. Taken together, the collection of NMR spectral data of MC modified protein samples offers a fertile source of information upon which a full map of solvent accessibility of the protein can be built.