Dataset Information

Probing residue-specific interactions in the stabilization of proteins using high-resolution NMR: a study of disulfide bond compensation.

ABSTRACT: It is well established that the oxidation state of cysteine residues in proteins is critical to overall physical stability. Disulfide bonds most often impart thermodynamic stability, but in some cases, diminish it. Predicting the circumstances that lead to each outcome is difficult because mechanistic information is lacking. Because the techniques typically used to study protein stability do not provide sufficient detail, high-resolution NMR was used in combination with low-resolution analysis to obtain mechanistic information regarding disulfide bond formation in a model protein. T(m) (CD) and T(onset) (SLS) for the reduced and oxidized wild type and C104S and C49S mutants were measured. The mutant proteins have altered T(m)s and T(onset)s compared to the reduced wild type, indicating that differences in local interactions of the Cys side chains are important for stability. The NMR spectra clearly show distinct differences in the chemical environment surrounding these Cys residues and the overall tertiary structure. The C49S protein, which is less stable and more aggregation prone than reduced wild type, lacks a hydrogen bond between Y53 and H103. Increased flexibility of the Y53-containing loop is correlated with increased dynamics and unraveling of alpha2, which likely leads to edge strand initiated aggregation of the central beta-sheet.

SUBMITTER: Skinner AL

PROVIDER: S-EPMC3683975 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Probing residue-specific interactions in the stabilization of proteins using high-resolution NMR: a study of disulfide bond compensation.

Skinner Andria L AL Laurence Jennifer S JS

Journal of pharmaceutical sciences 20100601 6

It is well established that the oxidation state of cysteine residues in proteins is critical to overall physical stability. Disulfide bonds most often impart thermodynamic stability, but in some cases, diminish it. Predicting the circumstances that lead to each outcome is difficult because mechanistic information is lacking. Because the techniques typically used to study protein stability do not provide sufficient detail, high-resolution NMR was used in combination with low-resolution analysis t ...[more]

PMID: 20187138

Similar Datasets

Project description:We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Dataset Information

Probing residue-specific interactions in the stabilization of proteins using high-resolution NMR: a study of disulfide bond compensation.

Publications

Probing residue-specific interactions in the stabilization of proteins using high-resolution NMR: a study of disulfide bond compensation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets