Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein.


Abstract

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to the prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-links on the unfolded state (chain entropy model) [Pace, C. N., Grimsley, G. R., Thomson, J. A., & Barnett, B. J. (1988) J. Biol. Chem. 263, 11820-11825: Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can influence the stability of the native state. In order to assess the importance of the latter effect, we have studied four variants of staphylococcal nuclease (V8 strain) each containing one potential disulfide bridge created by changing two wild-type residues to cysteines by site-directed mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro117, and the other was located in the adjacent extended loop containing Gly79. In all four cases, the overall loop size was kept nearly constant (the number of residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objective was to create variants in which a change in the reduction state of the disulfide would be coupled to a change in the position of the equilibrium between the cis and trans forms of the Xxx116-Pro117 peptide bond in the folded state of the protein. The position of this equilibrium, which can be detected by NMR spectroscopy, has been shown previously to correlate with the stability of the native protein. Its determination provides a measure of strain in the folded state. The thermal stabilities and free energies for unfolding by elevated temperature and guanidinium chloride were measured for each of the four mutants under conditions in which the introduced cysteines were cross-linked (oxidized) and unlinked (reduced). In addition, reduction potentials were determined for each mutant. Formation of the different disulfide bridges was found to induce varying levels of folded state strain. The stabilization energy of a given disulfide bridge could be predicted from the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native states of proteins may explain the variability observed in the stabilization provided by engineered disulfide bridges. Study holds ProTherm entries: 4808, 4809, 4810, 4811, 4812, 4813, 4814, 4815, 4816, 4817, 4818, 4819, 4820, 4821, 4822, 4823, 4824, 4825 Extra Details: chain entropy model; disulfide bridges; extended loop;,reduction state; stabilization energy

Submission Details

ID: v2Yak3qa3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Hinck AP;Truckses DM;Markley JL,Biochemistry (1996) Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein. PMID:8756688
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4WRD 2014-10-23T00:00:00+0000 1.65 Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
2LKV 2011-10-21T00:00:00+0000 0 Staphylococcal Nuclease PHS variant
2M00 2012-10-14T00:00:00+0000 0 Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+
2OXP 2007-02-20T00:00:00+0000 2.0 Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A
3D4W 2008-05-15T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature
3D8G 2008-05-23T00:00:00+0000 1.99 Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature
3MVV 2010-05-04T00:00:00+0000 1.72 Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature
3QOJ 2011-02-10T00:00:00+0000 1.6 Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K
3QOL 2011-02-10T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K
3R3O 2011-03-16T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.3 Thermonuclease Q6GIK1 NUC_STAAR
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.1 Thermonuclease Q7A6P2 NUC_STAAN
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.3 Thermonuclease Q5HHM4 NUC_STAAC
100.0 Thermonuclease P00644 NUC_STAAU