Contribution of salt bridges near the surface of a protein to the conformational stability.


Abstract

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl. Study holds ProTherm entries: 10238, 10239, 10240, 10241, 10242, 10243, 10244, 10245, 10246, 10247, 10248, 10249, 10250, 10251, 10266, 10267, 10268, 10269, 10270, 10271, 10272, 10273, 10274, 10275, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559 Extra Details: salt bridges; conformational stability; thermodynamic parameters;,solvent inaccessibility

Submission Details

ID: HLF69ceE4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:40 p.m.

Version: 1

Publication Details
Takano K;Tsuchimori K;Yamagata Y;Yutani K,Biochemistry (2000) Contribution of salt bridges near the surface of a protein to the conformational stability. PMID:11015217
Additional Information

Number of data points 84
Proteins Lysozyme C ; Lysozyme C
Unique complexes 7
Assays/Quantities/Protocols Experimental Assay: dG pH:4.5, buffers:sodium acetate: 0.02 M, ionic:KCl: 0.2 M ; Experimental Assay: dG pH:2.3, ionic:KCl: 0.2 M, buffers:glycine hydrochloride: 0.05 M ; Experimental Assay: dG buffers:sodium acetate: 0.02 M, ionic:-: -, pH:4.0 ; Experimental Assay: dG ionic:-: -, pH:2.2, buffers:glycine hydrochloride: 0.05 M ; Experimental Assay: Tm pH:4.5, buffers:sodium acetate: 0.02 M, ionic:KCl: 0.2 M ; Experimental Assay: Tm pH:2.3, ionic:KCl: 0.2 M, buffers:glycine hydrochloride: 0.05 M ; Experimental Assay: Tm pH:4.0, ionic:: , buffers:sodium acetate: 0.02 M ; Experimental Assay: Tm ionic:: , pH:2.2, buffers:glycine hydrochloride: 0.05 M ; Derived Quantity: ddG pH:4.5, buffers:sodium acetate: 0.02 M, ionic:KCl: 0.2 M ; Derived Quantity: ddG pH:2.3, ionic:KCl: 0.2 M, buffers:glycine hydrochloride: 0.05 M ; Derived Quantity: ddG buffers:sodium acetate: 0.02 M, ionic:-: -, pH:4.0 ; Derived Quantity: ddG ionic:-: -, pH:2.2, buffers:glycine hydrochloride: 0.05 M ; Derived Quantity: dTm pH:4.5, buffers:sodium acetate: 0.02 M, ionic:KCl: 0.2 M ; Derived Quantity: dTm pH:2.3, ionic:KCl: 0.2 M, buffers:glycine hydrochloride: 0.05 M ; Derived Quantity: dTm pH:4.0, ionic:: , buffers:sodium acetate: 0.02 M ; Derived Quantity: dTm ionic:: , pH:2.2, buffers:glycine hydrochloride: 0.05 M
Libraries Mutations for sequence KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV
Sequence Assay Result Units