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
2H45 2007-04-10 Solution structure of the second type III domain of human Fibronectin: ensemble of 25 structures
6HNF 2018-10-10 Structure in solution of human fibronectin type III-domain 14
2FN2 1998-09-16 SOLUTION NMR STRUCTURE OF THE GLYCOSYLATED SECOND TYPE TWO MODULE OF FIBRONECTIN, 20 STRUCTURES
5J6Z 2017-04-12 Structure of anastellin bound to beta-strands A and B from the third type III domain of fibronectin
2EC3 2008-03-04 Solution structure of the 11th FN1 domain from human Fibronectin 1
1E88 2000-10-09 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
2H41 2007-04-10 Solution structure of the second type III domain of human Fibronectin: minimized average structure
1TTG 1994-01-31 THE THREE-DIMENSIONAL STRUCTURE OF THE TENTH TYPE III MODULE OF FIBRONECTIN: AN INSIGHT INTO RGD-MEDIATED INTERACTIONS
1OWW 2003-10-07 Solution structure of the first type III module of human fibronectin determined by 1H, 15N NMR spectroscopy
1Q38 2003-11-04 Anastellin
1TTF 1994-01-31 THE THREE-DIMENSIONAL STRUCTURE OF THE TENTH TYPE III MODULE OF FIBRONECTIN: AN INSIGHT INTO RGD-MEDIATED INTERACTIONS
1FBR 1995-10-15 FOURTH AND FIFTH FIBRONECTIN TYPE I MODULE PAIR
2HA1 2007-04-10 Complex of the first and second type III domains of human Fibronectin in solution
2N1K 2015-11-11 Structure of the Third Type III Domain from Human Fibronectin
2MNU 2014-09-24 Backbone and side chain 1H, 13C, and 15N Chemical Shift Assignments for EDB and specific binding aptide
1E8B 2000-10-15 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
1O9A 2003-05-08 Solution structure of the complex of 1F12F1 from fibronectin with B3 from FnBB from S. dysgalactiae
1J8K 2002-02-06 NMR STRUCTURE OF THE FIBRONECTIN EDA DOMAIN, NMR, 20 STRUCTURES
2CKU 2007-04-03 Solution structure of 2F13F1 from human fibronectin
5M0A 2017-11-29 Solution structure of isolated 15th Fibronectin III domain from human fibronectin
2FNB 1998-12-23 NMR STRUCTURE OF THE FIBRONECTIN ED-B DOMAIN, NMR, 20 STRUCTURES
1QGB 1999-12-08 SOLUTION STRUCTURE OF THE N-TERMINAL F1 MODULE PAIR FROM HUMAN FIBRONECTIN
1QO6 2000-01-11 Solution structure of a pair of modules from the gelatin-binding domain of fibronectin
2CG7 2007-02-27 1.2 SECOND AND THIRD FIBRONECTIN TYPE I MODULE PAIR (CRYSTAL FORM II).
4LXO 2014-08-06 1.42 Crystal structure of 9,10Fn3-elegantin chimera
5DC4 2016-03-02 1.48 CRYSTAL STRUCTURE OF MONOBODY AS25/ABL1 SH2 DOMAIN COMPLEX, CRYSTAL A
2CG6 2007-02-27 1.55 Second and third fibronectin type I module pair (crystal form I).
5DC9 2016-03-02 1.56 CRYSTAL STRUCTURE OF MONOBODY AS25/ABL1 SH2 DOMAIN COMPLEX, CRYSTAL B
5N48 2018-02-14 1.6 Structure of Anticalin N9B in complex with extra-domain B of human oncofetal fibronectin
3CAL 2008-08-05 1.7 Crystal structure of the second and third fibronectin F1 modules in complex with a fragment of staphylococcus aureus fnbpa-5
3ZRZ 2011-09-28 1.7 Crystal structure of the second and third fibronectin F1 modules in complex with a fragment of Streptococcus pyogenes SfbI-5
1FNA 1994-04-30 1.8 CRYSTAL STRUCTURE OF THE TENTH TYPE III CELL ADHESION MODULE OF HUMAN FIBRONECTIN
2RKY 2008-08-05 1.8 Crystal structure of the fourth and fifth fibronectin F1 modules in complex with a fragment of staphylococcus aureus fnbpa-1
4PZ5 2014-07-02 1.96 Crystal structure of the second and third fibronectin F1 modules in complex with a fragment of BBK32 from Borrelia burgdorferi
2RKZ 2008-08-05 2.0 Crystal structure of the second and third fibronectin f1 modules in complex with a fragment of staphylococcus aureus fnbpa-1
2RL0 2008-08-05 2.0 Crystal structure of the fourth and fifth fibronectin F1 modules in complex with a fragment of staphylococcus aureus fnbpa-5
1FNF 1996-01-29 2.0 FRAGMENT OF HUMAN FIBRONECTIN ENCOMPASSING TYPE-III REPEATS 7 THROUGH 10
2CK2 2007-04-10 2.0 Structure of core-swapped mutant of fibronectin
2GEE 2006-10-17 2.01 Crystal Structure of Human Type III Fibronectin Extradomain B and Domain 8
3EJH 2009-02-03 2.1 Crystal Structure of the Fibronectin 8-9FnI Domain Pair in Complex with a Type-I Collagen Peptide
5DC0 2016-03-02 2.23 CRYSTAL STRUCTURE OF MONOBODY GG3/ABL1 SH2 DOMAIN COMPLEX
4JEG 2013-08-28 2.3 Crystal Structure of Monobody CS1/SHP2 C-SH2 Domain Complex
4JE4 2013-08-28 2.31 Crystal Structure of Monobody NSa1/SHP2 N-SH2 Domain Complex
3T1W 2012-03-21 2.4 Structure of the four-domain fragment Fn7B89 of oncofetal fibronectin
3R8Q 2012-04-11 2.4 Structure of Fibronectin domain 12-14
5DFT 2016-09-14 2.5 Structure of the Eleventh Type III Domain from Human Fibronectin
3M7P 2010-06-23 2.5 Fibronectin fragment
5J7C 2016-08-17 2.54 A picomolar affinity FN3 domain in complex with hen egg-white lysozyme
3GXE 2010-04-07 2.6 Complex of a Low Affinity Collagen Site with the Fibronectin 8-9FnI Domain Pair
4GH7 2012-12-26 2.6 Crystal structure of Anticalin N7A in complex with oncofetal fibronectin fragment Fn7B8
1FNH 1999-03-16 2.8 CRYSTAL STRUCTURE OF HEPARIN AND INTEGRIN BINDING SEGMENT OF HUMAN FIBRONECTIN
2OCF 2007-11-06 2.95 Human estrogen receptor alpha ligand-binding domain in complex with estradiol and the E2#23 FN3 monobody
3MQL 2010-08-25 3.0 Crystal structure of the fibronectin 6FnI1-2FnII7FnI fragment
5N47 2018-02-14 3.0 Structure of Anticalin N7E in complex with the three-domain fragment Fn7B8 of human oncofetal fibronectin
4MMZ 2014-03-26 3.1 Integrin AlphaVBeta3 ectodomain bound to an antagonistic tenth domain of Fibronectin
4MMY 2014-03-26 3.18 Integrin AlphaVBeta3 ectodomain bound to the tenth domain of Fibronectin with the IAKGDWND motif
4MMX 2014-03-26 3.32 Integrin AlphaVBeta3 ectodomain bound to the tenth domain of Fibronectin

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