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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling
Thursday Speaker Abstracts
Scanning Protein Surface with a Solvent Mimetic Probe: an NMR Approach
Gabriela E. Gomez
1
, Evangelina M. Bernar
1
, Martin Aran
2
,
Jose M. Delfino
1
.
1
Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires,
Argentina,
2
Fundacion Instituto Leloir, Buenos Aires, Argentina.
Changes in the solvent accessible surface area (SASA) of proteins underlie protein folding,
molecular recognition phenomena, and assembly of complexes. Nevertheless, this fundamental
parameter becomes elusive for experimental scrutiny. Methylene carbene (MC) reacts with
polypeptides allowing an estimate of the magnitude and nature of SASA. MC arises from the
photocleavage of diazirine (DZN), a tiny heterocycle similar in size and shape to water, thus able
to exert solvent mimicry. MC is an extremely reactive species that inserts readily into X-H bonds
(X=C, O, S or N). Coupled to radioactivity or mass spectrometry detection, DZN labeling
becomes useful to probe folding and interactions. By contrast, multidimensional NMR does not
demand cleavage of the polypeptide, potentially opening a rich panorama on structure. General
methylation was assessed in E. coli thioredoxin (TRX), where the insertion of MC at multiple
sites across the surface was verified. 1H-NMR spectra of progressively reacted TRX show a
significant enrichment in the aliphatic region, supporting the dominant insertion event into C-H
bonds belonging to amino acid side-chains. Remarkably, buried residues such as Val 16 remain
unmodified. Consistently, 1H,13C-HSQC spectra uncover new cross-peaks corresponding to
water exposed methyl groups, while concurrently protein methylene signals disappear. On the
other hand, 1H,15N-HSQC spectra reveal the impact of side-chain methylation on backbone
amide environments. Discrete alterations occur at indole groups of partially exposed Trp 28 and
31. By contrast, backbone methylation itself is a rare occurrence. Because of its mild reaction
conditions and strong focus on side-chain modification, DZN labeling adds its unique value to
current footprinting methods, such as H/D exchange and hydroxyl radical reactions. Collectively,
NMR analysis of MC modified proteins offers a fertile source of information upon which a full
map of solvent accessibility can be built.