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Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease
D M Truckses1, J R Somoza, K E Prehoda
1Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706, USA.
Protein Science : a Publication of the Protein Society
|September 1, 1996
Summary
Protein stability differences in Staphylococcus aureus nucleases are linked to amino acid variations at residue 124. These changes affect the conformation of a nearby peptide bond, influencing overall protein structure and function.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Engineering
Background:
- Staphylococcus aureus nuclease variants (WT with His124, H124L with Leu124) exhibit differing stabilities.
- A single amino acid substitution at residue 124 significantly impacts nuclease stability and peptide bond conformation.
Purpose of the Study:
- To elucidate the molecular basis for stability differences between Staphylococcus aureus nuclease variants.
- To investigate the correlation between residue 124 identity, protein stability, and peptide bond cis/trans isomerization.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to analyze peptide bond conformation.
- X-ray crystallography to determine protein structures.
- Site-directed mutagenesis to create variants (H124A, H124I, P117G, P47G).
- Enzyme kinetics assays (kcat, Km) to assess nuclease activity.
Main Results:
- A correlation was found between residue 124 substitutions, increased protein stability, and stabilization of the cis configuration at the Lys116-Pro117 peptide bond.
- X-ray structures of H124L and WT nucleases revealed no significant structural differences beyond residue 124.
- Proline to glycine substitutions at residues 117 or 47 increased protein stability and altered local loop conformations.
- Mutations P117G and P47G decreased nuclease activity, with additive effects.
Conclusions:
- Stability differences in Staphylococcus aureus nuclease variants primarily arise from subtle structural changes affecting the cis/trans equilibrium of the Lys116-Pro117 peptide bond.
- The observed stability changes are not solely attributable to helical propensity differences.
- Proline substitutions at positions 47 and 117 can modulate nuclease stability and activity through conformational rearrangements.