Importance of the C-Terminal Loop L137−S141 for the Folding and Folding Stability of Staphylococcal Nuclease^,

Abstract

The role of the C-terminal loop L137-S141 in the folding and folding stability of staphylococcal nuclease (SNase) was investigated by deletion mutation. The C-terminal truncated SNase fragments, SNase137, SNase139, SNase140, and SNase141 containing residues 1-137, 1-139, 1-140, and 1-141, respectively, were adopted in this study. Folding states of these four SNase fragments were analyzed by circular dichroism and fluorescence measurements. The solution structure of SNase140 was determined and compared to those of SNase141 and native SNase using the heteronuclear NMR method. The results showed that folding of the four SNase fragments is correlated with the folding of helix R3. With the chain length extending from L137 and I139 to S141, folding of the fragments progressively approached to the tertiary folding of native SNase, and the folding stability was enhanced. These observations revealed that the C-terminal loop L137-S141 has profound effect not only on the folding of helix R3 but also on the stabilizing folding of both the R-and β-subdomains of SNase. Analysis indicates that stabilizing folding of the SNase and SNase fragments depends to a large extent on the hydrophobic packing interactions in both the C-terminal local structural region surrounding W140 including the loop L137-S141 and the N-terminal local structural region of the "β-barrel" hydrophobic core.Over the past years, a number of studies have been carried out on addressing the information necessary for stabilizing the folding of staphylococcal nuclease (SNase) 1 encoded by the protein sequence (1-6). SNase is a widely used model system for the study of protein folding, which is composed of two subdomains, a large N-terminal β-subdomain and a small C-terminal R-subdomain. The N-terminal β-subdomain is constructed by a "β-barrel" core and two helices R1 (A58-A69) and R2 (V99-Q106) as well as the p-loop (pdTp-binding loop, D77-L89) and Ω-loop (P42-P56), whereas the C-terminal R-subdomain contains mainly a helix R3 (E122-K136) and the subsequent helical turn L137-S141 and the N-and C-terminal loops of the subdomain (7,8). A mixture of the N-terminal β-subdomain fragment and the C-terminal R-subdomain fragment can form a noncovalent complex through a folding-onbinding event, which generates a tertiary structure nearly identical to the structure of native SNase. For this, the crucial elements in the surface region of the binding interface between two subdomains are required, which establish the interactions necessary for stabilizing the native-like folding of both fragments (8). The C-terminal amino acids 137-149 of SNase were proposed to contain critical information for the native folding of SNase (1, 2). The 3D solution structure of full-length SNase shows that the loop L137-S141 connecting to the C-terminal end of the helix R3 has a helical-turn conformation (7). W140 in this loop region is a key residue which bears information important to both long-range and short-range interactions for the structure and stability of SNase and plays an i...

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.…”

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…It is not surprising that the 10-23 DNAzyme has the highest activity with Pb 2+ since both DNAzymes share the same conserved nucleotides. 31 If we compare both, the 8-17 DNAzyme exhibits a similar trend but is ~100-fold more active at the same metal concentrations ( Figure 1F, note the difference in the scale of the y-axis). The significance of the similar trend observed in the above experiment also lies in the fact that it supports the theory that these two DNAzymes might belong to the same family and undergo the similar cleavage mechanism.…”

“…It was recently proposed that the 8-17 and 10-23 DNAzymes might be the same enzyme. 31 In other words, the 10-23 DNAzyme might be a mutant of the 8-17. 31 This conclusion was made based on mutation studies.…”

“…31 In other words, the 10-23 DNAzyme might be a mutant of the 8-17. 31 This conclusion was made based on mutation studies. We are interested in testing their activity in the presence of different metal ions.…”

CING: an integrated residue-based structure validation program suite

“…2 for examples) show significant variations in the allowed regions and distributions of the different secondary structural elements, colour coded as in the Ramachandran and Janin plots.

Fig. 2Overview of the CING analysis for PDB entry 2kq3 (Wang et al 2010). a Project page of entry 2kq3.

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