Lysines in the amino-terminal alpha-helix are important to the stability of Rhodobacter capsulatus cytochrome c2.


Abstract

The protein stabilities of wild type and four site-directed mutants of Rhodobacter capsulatus cytochrome c2 have been characterized. The integrity of the cytochrome c2 iron-sulfur environment was ascertained by titration of the 696-nm absorbance band with alkali, and the conformational stability was determined by titration of the 220-nm circular dichroism signal with Gdn-HCl. Analysis of the alkaline transition pK value of K12D (lysine-12 substituted by aspartate) indicated that the K12D iron-sulfur environment was destabilized by 0.6 kcal/mol relative to the wild-type cytochrome c2 at low ionic strength. In contrast, the alkaline transition pK values of K14E (lysine-14 substituted by glutamate), K32E (lysine-32 substituted by glutamate), and K14E/K32E (lysines-14 and -32 substituted by glutamates) were indistinguishable from the wild type, indicating that these substitutions have no effect on the stability of the iron-sulfur environment. Gdn-HCl denaturation of K12D and K14E indicated that both these mutations decreased conformational stability by 1.3 kcal/mol. In contrast, mutant K32E exhibited a small stabilizing effect of 0.2 kcal/mol. Gdn-HCl denaturation of K14E/K32E indicated that this mutation decreased conformational stability by 1.3 kcal/mol, which is consistent with the additive effects of the single charge mutations at positions 14 and 32. The conformational instability of mutants possessing negative charges at position 12 or 14 is best explained by their positioning at the carboxy-terminal region of the amino-terminal alpha-helix of R. capsulatus cytochrome c2. Accordingly, introduction of negatively charged groups into this region appears to destabilize cytochrome c2 through energetically unfavorable interactions with the dipole of the amino-terminal helix.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 351, 352, 353, 354, 2210 Extra Details: dG and ddG were measured in the presence of [GdnHCl]50% Rhodobacter capsulatus cytochrome c2; alpha-helix; circular dichroism;,conformational stability; dipole

Submission Details

ID: svKVPU4F3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Caffrey MS;Cusanovich MA,Biochemistry (1991) Lysines in the amino-terminal alpha-helix are important to the stability of Rhodobacter capsulatus cytochrome c2. PMID:1654091
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
1C2N 1999-03-23 CYTOCHROME C2, NMR, 20 STRUCTURES
1VYD 2004-06-17 2.3 Crystal structure of cytochrome C2 mutant G95E
1C2R 1992-01-15 2.5 MOLECULAR STRUCTURE OF CYTOCHROME C2 ISOLATED FROM RHODOBACTER CAPSULATUS DETERMINED AT 2.5 ANGSTROMS RESOLUTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cytochrome c2 P00094 CYC2_RHOCB