Surface point mutations that significantly alter the structure and stability of a protein's denatured state.


Abstract

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

Submission Details

ID: Mr2ScbrD4

Submitter: Connie Wang

Submission Date: Aug. 1, 2017, 12:31 p.m.

Version: 1

Publication Details
Smith CK;Bu Z;Anderson KS;Sturtevant JM;Engelman DM;Regan L,Protein Sci (1996) Surface point mutations that significantly alter the structure and stability of a protein's denatured state. PMID:8897601
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
2IGH 1994-01-31 DETERMINATION OF THE SOLUTION STRUCTURES OF DOMAINS II AND III OF PROTEIN G FROM STREPTOCOCCUS BY 1H NMR
2PLP 2007-05-08 Ultra high resolution backbone conformation of protein GB1 from residual dipolar couplings alone
2KBT 2009-02-03 Attachment of an NMR-invisible solubility enhancement tag (INSET) using a sortase-mediated protein ligation method
1GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
2LGI 2011-10-26 Atomic Resolution Protein Structures using NMR Chemical Shift Tensors
1Q10 2003-10-14 Ensemble of 40 Structures of the Dimeric Mutant of the B1 Domain of Streptococcal Protein G
2MBB 2014-06-04 Solution Structure of the human Polymerase iota UBM1-Ubiquitin Complex
2K0P 2009-03-03 Determination of a Protein Structure in the Solid State from NMR Chemical Shifts
2NMQ 2006-11-21 Simultaneous determination of protein structure and dynamics using rdcs
2N7J 2015-10-14 Sidechain chi1 distribution in B3 domain of protein G from extensive sets of residual dipolar couplings
2RPV 2009-09-15 Solution Structure of GB1 with LBT probe
2J53 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
1PN5 2003-10-07 NMR structure of the NALP1 Pyrin domain (PYD)
2RMM 2007-12-04 Solution structure of GB1 A34F mutant
1LE3 2002-04-24 NMR Structure of Tryptophan Zipper 4: A Stable Beta-Hairpin Peptide Based on the C-terminal Hairpin of the B1 Domain of Protein G
2KLK 2009-10-06 Solution structure of GB1 A34F mutant with RDC and SAXS
2GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
3GB1 1999-06-23 STRUCTURES OF B1 DOMAIN OF STREPTOCOCCAL PROTEIN G
2J52 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
1MPE 2002-10-30 Ensemble of 20 structures of the tetrameric mutant of the B1 domain of streptococcal protein G
2IGD 1998-07-29 1.1 ANISOTROPIC STRUCTURE OF PROTEIN G IGG-BINDING DOMAIN III AT 1.1 ANGSTROM RESOLUTION
1IGD 1994-11-01 1.1 THE THIRD IGG-BINDING DOMAIN FROM STREPTOCOCCAL PROTEIN G: AN ANALYSIS BY X-RAY CRYSTALLOGRAPHY OF THE STRUCTURE ALONE AND IN A COMPLEX WITH FAB
3MP9 2011-02-23 1.2 Structure of Streptococcal protein G B1 domain at pH 3.0
6CNE 2019-07-10 1.2 Selenomethionine mutant (L5Sem) of protein GB1 examined by X-ray diffraction
1PGX 1992-07-15 1.66 THE 1.66 ANGSTROMS X-RAY STRUCTURE OF THE B2 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON TO THE NMR STRUCTURE OF THE B1 DOMAIN
1PGB 1994-04-30 1.92 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCCOCAL PROTEIN G AND COMPARISON WITH NMR
4KGS 2013-09-04 1.95 Backbone Modifications in the Protein GB1 Loops: beta-3-Val21, beta-3-Asp40
4KGT 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Turns: Aib10, D-Pro47
1EM7 2002-05-08 2.0 HELIX VARIANT OF THE B1 DOMAIN FROM STREPTOCOCCAL PROTEIN G
4KGR 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Helix: beta-3-Ala24, beta-3-Lys28, beta-3-Lys31, beta-3-Asn35
1PGA 1994-04-30 2.07 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON WITH NMR
1MVK 2002-10-30 2.5 X-ray structure of the tetrameric mutant of the B1 domain of streptococcal protein G
1IGC 1995-06-03 2.6 IGG1 FAB FRAGMENT (MOPC21) COMPLEX WITH DOMAIN III OF PROTEIN G FROM STREPTOCOCCUS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Immunoglobulin G-binding protein G P06654 SPG1_STRSG