Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

Ye, Keqiong; Wang, Jinfeng

doi:10.1006/jmbi.2000.4449

Cited by 7 publications

(4 citation statements)

References 47 publications

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“…It is of interest therefore to explore the possibility that partially folded intermediates of spc-SH3 might become stabilized by intermolecular association favored by the high concentrations used in the NMR experiments. The self-association of folding intermediates and denatured states of proteins and protein fragments has been reported previously (Filimonov et al, 1993;Pecorari et al, 1996;Uversky et al, 1998;Kuznetsova et al, 1999;Liu et al, 1999;Uversky et al, 1999;Ye and Wang, 2001). The oligomeric states have been often described as an association of folding intermediates with residual super-secondary structure (Filimonov et al, 1993), which in certain cases appears as native-like (Alexandrescu et al, 2000;Ye and Wang, 2001).…”

Section: Introductionsupporting

confidence: 52%

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

Casares

Sadqi

López-Mayorga

et al. 2004

Biophysical Journal

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For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

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Section: Introductionsupporting

confidence: 52%

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

Casares

Sadqi

López-Mayorga

et al. 2004

Biophysical Journal

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“…A table of these values is provided in the Supporting Information. The values of R 2 can be increased by conformational exchange due to self-association (35). The R 2 value at 600 MHz was estimated for five protein concentrations ranging from 0.05 to 0.4 mM in determining the contribution to R 2 from self-association.…”

Section: N Relaxationmentioning

confidence: 99%

Backbone Dynamics of the Monomeric λ Repressor Denatured State Ensemble under Nondenaturing Conditions

Chugha

Oas

2007

Biochemistry

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Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.

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“…We initially looked at residual structure under strongly denaturing conditions. An interesting observation to emerge from this work is that the aggregation of CspA, LysN, and SN under acid denaturing conditions is coupled to the propensity of the OB-fold β-meander (cyan in Figure 1B) to adopt residual structure in the unfolded states (39,50,59,60). Subsequently, it has been shown that the uncoupling of the β-meander from the rest of the CspA structure might have a functional role in nucleic acid binding (61) and that the β-meander of CspA can act as a template to fold unrelated sequences in a chimera model of protein structure evolution (62).…”

Section: Discussionmentioning

confidence: 87%

“…It has been suggested that the flexibility of peripheral structures within an otherwise conserved fold allows for structural diversity, for example, the genesis of new structures through oligomerization (64). There is support for this hypothesis from the OB-fold where the relative independence of the β1-3 meander leads to the apparently conserved aggregation of the acid-denatured states of all three proteins (39,50,60). Moreover, perturbation of the loop between the nonconserved α-helices 1 and 2 in SN results in a highly stable domain-swapped dimer in which the OB-fold is structurally conserved, and helix α2 swaps from one monomer to the other (65).…”

Section: Discussionmentioning

confidence: 99%

Partially Folded States of Staphylococcal Nuclease Highlight the Conserved Structural Hierarchy of OB-Fold Proteins

et al. 2007

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We have been interested in whether three proteins that share a five-stranded β-barrel "OB-fold" structural motif but no detectable sequence homology fold by similar mechanisms. Here we describe native-state hydrogen exchange experiments as a function of urea for SN (staphylococcal nuclease), a protein with an OB-fold motif and additional nonconserved elements of structure. The regions of structure with the largest stability and unfolding cooperativity are contained within the conserved OB-fold portion of SN, consistent with previous results for CspA (cold shock protein A) and LysN (anticodon binding domain of lysyl tRNA synthetase). The OB-fold also has the subset of residues with the slowest unfolding rates in the three proteins, as determined by hydrogen exchange experiments in the EX1 limit. Although the protein folding hierarchy is maintained at the level of supersecondary structure, it is not evident for individual residues as might be expected if folding depended on obligatory nucleation sites. Rather, the site-specific stability profiles appear to be linked to sequence hydrophobicity and to the density of long-range contacts at each site in the threedimensional structures of the proteins. We discuss the implications of the correlation between stability to unfolding and conservation of structure for mechanisms of protein structure evolution.The concept of a protein fold as a recurrent unit of structure is central to modern structural biology. One of the goals of The Protein Structure Initiative is to determine representative examples of all folds (1), estimated to number between 1000 (2) and 10 000 (1,3,4). The difficulty of protein structure prediction targets and the approaches for modeling unknown structures are often predicated on whether the protein is likely to have a novel fold (5). Inferences about evolutionary relationships are often made on the basis of protein folds, because structure can be conserved even when sequences have diverged to levels where sequence homology is no longer detectable (6). While the concept of a protein fold is generally accepted, the classification of folds in practice remains somewhat subjective and ambiguous.The OB-fold 1 (Figure 1) was originally described as an oligonucleotide/oligosaccharidebinding fold (7) and consists of a five-stranded β-barrel structure. It is represented well in both sequence and structure databases (3,(8)(9)(10). The October 2006 release of the SCOP database (11) classifies some 95 protein domains as OB-fold structures. These are grouped into 27 function families with activities ranging from molybdate-binding proteins to RNA chaperones. † This work was supported by NSF Grant MB 0236316 to A.T.A., a Richard Crain Jr. Memorial Fellowship to W.M.M., and a Todd M.Schuster undergraduate summer fellowship to E.W. * To whom correspondence should be addressed: Department of Molecular and Cell Biology, University of Connecticut, 91 N,. Eagleville Rd., U-3125, Storrs, CT 06269-3125., Telephone: (860) Fax: (860) ., E-mail: andrei@uconn.edu.. ‡...

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Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

Cited by 7 publications

References 47 publications

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

Backbone Dynamics of the Monomeric λ Repressor Denatured State Ensemble under Nondenaturing Conditions

Partially Folded States of Staphylococcal Nuclease Highlight the Conserved Structural Hierarchy of OB-Fold Proteins

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