Thermodynamics of the unfolding and spectroscopic properties of the V66W mutant of Staphylococcal nuclease and its 1-136 fragment.


Abstract

Spectroscopic studies have been performed to characterize the solution structure of the V66W mutant of Staphylococcal nuclease and the corresponding 1-136 fragment, referred to as V66W'. Whereas wild-type nuclease has a single tryptophan residue at position 140, the V66W mutant has a second tryptophan residue at position 66, which is the only such residue in V66W'. Steady-state and time-resolved fluorescence studies show Trp-66 in V66W' to have a blue emission, a relatively large fluorescence quantum yield, a long lifetime, a significant degree of protection from solute quenchers, and to depolarize with a relatively long rotational correlation time. These results characterize Trp-66 in V66W' as being a buried residue, which indicates that this fragment retains some global structure. Circular dichroism (CD) data are consistent with the fragment having lost most of the alpha-helical content of the wild type, while retaining beta-sheet structure. The CD spectrum in the aromatic region also suggests that Trp-66 in the fragment experiences an asymmetric environment, which is not identical to that in the full length mutant, V66W. In addition, optical detection of triplet state magnetic resonance (ODMR) spectroscopy can clearly resolve the tryptophan residues and demonstrates differences between the local environment of Trp-66 in V66W and in V66W', as well as small differences in the Trp-140 environment in wild type and in V66W. Guanidine-HCl induced and thermally induced unfolding studies were performed by simultaneously acquiring CD and fluorescence data as a function of the perturbation and then performing a global analysis of such multiple data sets in terms of two-state and three-state unfolding models. Whereas data for wild-type nuclease and the V66W' fragment are well characterized by a two-state unfolding model, data for the V66W mutant are better characterized by a three-state process. That is, both the denaturant- and temperature-induced unfolding of V66W involves the significant population of an equilibrium unfolding intermediate. Our global analyses yield thermodynamic parameters for the unfolding transitions, and we show that the data for V66W can be described by a constrained three-state model in which the transition of the intermediate to the fully unfolded state is fixed to have the same thermodynamic parameters that describe the unfolding of the V66W' fragment. Study holds ProTherm entries: 4678, 4679, 4680, 4681 Extra Details: correlation time; buried residue; global structure;,alpha-helical content; beta-sheet structure; local environment

Submission Details

ID: 4L6D8Kgt3

Submitter: Connie Wang

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

Version: 1

Publication Details
Eftink MR;Ionescu R;Ramsay GD;Wong CY;Wu JQ;Maki AH,Biochemistry (1996) Thermodynamics of the unfolding and spectroscopic properties of the V66W mutant of Staphylococcal nuclease and its 1-136 fragment. PMID:8672513
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