Rational modification of protein stability by the mutation of charged surface residues.


Abstract

Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 degrees C, the T(m) of the wild-type, of 0.86 and 1.08 kcal mol(-)(1), respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein. Study holds ProTherm entries: 8036 Extra Details: peripheral subunit-binding domain; electrostatic potential;,isosteric; surface charge

Submission Details

ID: UUiCnb7T4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Spector S;Wang M;Carp SA;Robblee J;Hendsch ZS;Fairman R;Tidor B;Raleigh DP,Biochemistry (2000) Rational modification of protein stability by the mutation of charged surface residues. PMID:10653630
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
1W4F 2005-07-20 Peripheral-subunit from mesophilic, thermophilic and hyperthermophilic bacteria fold by ultrafast, apparently two-state transitions
1LAC 1993-07-15 THREE-DIMENSIONAL STRUCTURE OF THE LIPOYL DOMAIN FROM BACILLUS STEAROTHERMOPHILUS PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX
2PDD 1994-12-20 THE HIGH RESOLUTION STRUCTURE OF THE PERIPHERAL SUBUNIT-BINDING DOMAIN OF DIHYDROLIPOAMIDE ACETYLTRANSFERASE FROM THE PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX OF BACILLUS STEAROTHERMOPHILUS
1W3D 2004-07-20 NMR structure of the peripheral-subunit binding domain of Bacillus stearothermophilus E2p
1LAB 1993-07-15 THREE-DIMENSIONAL STRUCTURE OF THE LIPOYL DOMAIN FROM BACILLUS STEAROTHERMOPHILUS PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX
1W4E 2005-07-20 Peripheral-subunit binding domains from mesophilic, thermophilic, and hyperthermophilic bacteria fold by ultrafast, apparently two-state transitions
1W4G 2005-07-20 Peripheral-subunit binding domains from mesophilic, thermophilic, and hyperthermophilic bacteria fold by ultrafast, apparently two-state folding transitions
2PDE 1994-12-20 THE HIGH RESOLUTION STRUCTURE OF THE PERIPHERAL SUBUNIT-BINDING DOMAIN OF DIHYDROLIPOAMIDE ACETYLTRANSFERASE FROM THE PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX OF BACILLUS STEAROTHERMOPHILUS
1W85 2004-11-02 2.0 The crystal structure of pyruvate dehydrogenase E1 bound to the peripheral subunit binding domain of E2
1W88 2004-11-02 2.3 The crystal structure of pyruvate dehydrogenase E1(D180N,E183Q) bound to the peripheral subunit binding domain of E2
3DVA 2009-01-13 2.35 Snapshots of catalysis in the E1 subunit of the pyruvate dehydrogenase multi-enzyme complex
3DUF 2009-01-06 2.5 Snapshots of catalysis in the E1 subunit of the pyruvate dehydrogenase multi-enzyme complex
3DV0 2009-01-13 2.5 Snapshots of catalysis in the E1 subunit of the pyruvate dehydrogenase multi-enzyme complex
1EBD 1996-07-11 2.6 DIHYDROLIPOAMIDE DEHYDROGENASE COMPLEXED WITH THE BINDING DOMAIN OF THE DIHYDROLIPOAMIDE ACETYLASE
1B5S 1999-02-16 4.4 DIHYDROLIPOYL TRANSACETYLASE (E.C.2.3.1.12) CATALYTIC DOMAIN (RESIDUES 184-425) FROM BACILLUS STEAROTHERMOPHILUS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex P11961 ODP2_GEOSE