Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface.


Abstract

It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions. Study holds ProTherm entries: 11625, 11626, 11627, 11628, 11629, 11630, 11631, 11632, 11633, 11634, 11635, 11636 Extra Details: in the presence of 6.3 M Urea electrostatic interactions; protein surface; ion pairs; negatively charged patch

Submission Details

ID: WhjDk9UZ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
Koide A;Jordan MR;Horner SR;Batori V;Koide S,Biochemistry (2001) Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface. PMID:11513611
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
1E88 2000-09-18T00:00:00+0000 0 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
1E8B 2000-09-18T00:00:00+0000 0 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
1FBR 1995-08-08T00:00:00+0000 0 FOURTH AND FIFTH FIBRONECTIN TYPE I MODULE PAIR
1FNA 1994-01-11T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF THE TENTH TYPE III CELL ADHESION MODULE OF HUMAN FIBRONECTIN
1FNF 1995-09-30T00:00:00+0000 2.0 FRAGMENT OF HUMAN FIBRONECTIN ENCOMPASSING TYPE-III REPEATS 7 THROUGH 10
1FNH 1999-01-28T00:00:00+0000 2.8 CRYSTAL STRUCTURE OF HEPARIN AND INTEGRIN BINDING SEGMENT OF HUMAN FIBRONECTIN
1J8K 2001-05-22T00:00:00+0000 0 NMR STRUCTURE OF THE FIBRONECTIN EDA DOMAIN, NMR, 20 STRUCTURES
1O9A 2002-12-11T00:00:00+0000 0 Solution structure of the complex of 1F12F1 from fibronectin with B3 from FnBB from S. dysgalactiae
1OWW 2003-03-31T00:00:00+0000 0 Solution structure of the first type III module of human fibronectin determined by 1H, 15N NMR spectroscopy
1Q38 2003-07-28T00:00:00+0000 0 Anastellin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.8 Fibronectin P07589 FINC_BOVIN
100.0 Fibronectin P02751 FINC_HUMAN
90.9 Fibronectin Q91400 FINC_NOTVI
92.1 Fibronectin Q28377 FINC_HORSE
94.0 Fibronectin Q28275 FINC_CANLF