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

Abstract

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.. ‡...

Extracting function from a β-trefoil folding motif

“…Function evolves in proteins through the acquisition of surface loops (48), and such loops are believed to not affect protein stability (49) and folding. Our results indicate that protein structure, function, and folding are inextricably related and that the structural differences between structurally similar proteins correlate not only with functional regions but also with topological folding traps.…”

Amide Proton Solvent Protection in Amylin Fibrils Probed by Quenched Hydrogen Exchange NMR

“…The observed differences in exchange rates between residues within the same strand (e.g. G33 and Y37 from strand β2), suggests that structural stability varies within a given element of secondary structure, as is often found in folded globular proteins [17], [34].…”

“…The GNM formalism models fluctuations about a mean structure as dependent on the distribution of distance contacts to nearby Cα atoms [42]. The predicted amplitudes of fluctuations at different sites can be used to calculate theoretical B-factors [42], which for native proteins have been shown to be in good agreement with experimental B-factors determined by X-ray crystallography and to correlate with HX protection factors [34], [42]–[44]. The theoretical B-factors calculated for the amylin fibril model are shown by the black symbols in Fig.…”

Cavities determine the pressure unfolding of proteins

“…Beyond such observations, by focusing on the pressure unfolding reaction proper, we have characterized, with unprecedented structural and energetic resolution, the pressure unfolding landscape of SNase and its variants. Deletion mutagenesis (33), H/D exchange (34,35), and mechanical unfolding experiments (36) all at atmospheric pressure have provided a consistent picture of the modular nature of SNase with two subdomains, an N-terminal β-barrel core domain that includes helix 1, and a C-terminal domain consisting primarily of helix 3, with helix 2 acting as an interface between the two subdomains and the C-terminal loop Leu-137 to Ser-141 locking the protein into its correct tertiary structure through long range contacts (37,38). Besides the typical cis/trans proline isomerization observed within the unfolded states on a slow time scale, multiphasic kinetics have been detected in a stopped-flow study of a proline-free variant of SNase (39), suggesting the accumulation of on-pathway intermediate states.…”