Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects.


Abstract

Electrostatic contributions to the conformational stability of apoflavodoxin were studied by measurement of the proton and salt-linked stability of this highly acidic protein with urea and temperature denaturation. Structure-based calculations of electrostatic Gibbs free energy were performed in parallel over a range of pH values and salt concentrations with an empirical continuum method. The stability of apoflavodoxin was higher near the isoelectric point (pH 4) than at neutral pH. This behavior was captured quantitatively by the structure-based calculations. In addition, the calculations showed that increasing salt concentration in the range of 0 to 500 mM stabilized the protein, which was confirmed experimentally. The effects of salts on stability were strongly dependent on cationic species: K(+), Na(+), Ca(2+), and Mg(2+) exerted similar effects, much different from the effect measured in the presence of the bulky choline cation. Thus cations bind weakly to the negatively charged surface of apoflavodoxin. The similar magnitude of the effects exerted by different cations indicates that their hydration shells are not disrupted significantly by interactions with the protein. Site-directed mutagenesis of selected residues and the analysis of truncation variants indicate that cation binding is not site-specific and that the cation-binding regions are located in the central region of the protein sequence. Three-state analysis of the thermal denaturation indicates that the equilibrium intermediate populated during thermal unfolding is competent to bind cations. The unusual increase in the stability of apoflavodoxin at neutral pH affected by salts is likely to be a common property among highly acidic proteins. Study holds ProTherm entries: 14795, 14796, 14797, 14798, 14799, 14800, 14801, 14802, 14803, 14804, 14805, 14806 Extra Details: electrostatics; protein stability; flavodoxin; ion binding; ionic,strength; cations; protein folding

Submission Details

ID: 57HmTysG

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Maldonado S;Irún MP;Campos LA;Rubio JA;Luquita A;Lostao A;Wang R;García-Moreno E B;Sancho J,Protein Sci. (2002) Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects. PMID:11967382
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
2KQU 2010-06-02 F98N apoflavodoxin from Anabaena PCC 7119
5LJP 2017-08-02 1.1 E20K/I59A/E72K/I92A/D126K/A142V FLAVODOXIN FROM ANABAENA
1OBO 2003-04-24 1.2 W57L flavodoxin from Anabaena
1RCF 1995-01-26 1.4 STRUCTURE OF THE TRIGONAL FORM OF RECOMBINANT OXIDIZED FLAVODOXIN FROM ANABAENA 7120 AT 1.40 ANGSTROMS RESOLUTION
2V5V 2007-10-16 1.88 W57E Flavodoxin from Anabaena
3ESZ 2009-02-10 1.94 K2AK3A Flavodoxin from Anabaena
2V5U 2007-10-16 1.99 I92A FLAVODOXIN FROM ANABAENA
1FTG 1996-12-23 2.0 STRUCTURE OF APOFLAVODOXIN: CLOSURE OF A TYROSINE/TRYPTOPHAN AROMATIC GATE LEADS TO A COMPACT FOLD
1QHE 1999-05-20 2.0 ENERGETICS OF A HYDROGEN BOND (CHARGED AND NEUTRAL) AND OF A CATION-PI INTERACTION IN APOFLAVODOXIN
1FLV 1993-10-31 2.0 STRUCTURE OF THE OXIDIZED LONG CHAIN FLAVODOXIN FROM ANABAENA 7120 AT 2 ANGSTROMS RESOLUTION
1DX9 2000-04-10 2.05 W57A Apoflavodoxin from Anabaena
1OBV 2003-04-24 2.1 Y94F flavodoxin from Anabaena
3ESX 2009-02-10 2.31 E16KE61KD126KD150K Flavodoxin from Anabaena
3ESY 2009-02-10 2.39 E16KE61K Flavodoxin from Anabaena

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Flavodoxin P0A3E0 FLAV_NOSSO
100.0 Flavodoxin P0A3D9 FLAV_NOSS1