Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies.


Abstract

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41-->Val and Leu-41-->Ala) in the dimeric 4-alpha-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in alpha-helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. delta H zero values at the respective transition temperatures, T 1/2 (71.0, 65.3, and 52.9 degrees C at 0.5 mg/ml) decrease from 580 +/- 20 to 461 +/- 20 kJ/(mol of dimer) and 335 +/- 20 kJ/(mol of dimer) for wild-type ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L41V, and L41A, respectively. The conformational stabilities at 25 degrees C expressed by the standard Gibbs energies of denaturation, delta GzeroD, are 71.7, 61.1, and 46.1 kJ/(mol of dimer). The corresponding transition enthalpies have been obtained from extrapolation using the cDp(T) and cNp(T) functions. Their values at 25 degrees C are 176.3, 101.9, and 141.7 kJ/(mol of dimer) for wild-type ROP, L41V, and L41A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH2 group, the average delta delta GzeroD value is -5.0 +/- 1 kJ/(mol of CH2 group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu-->Val and Leu-->Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state. Study holds ProTherm entries: 3104, 3105, 3106, 11859, 11860, 11861 Extra Details: additive : EDTA(1 mM) protein stability; 4-alpha-helix bundle; ROP protein;,cavity mutations; heat capacity

Submission Details

ID: Bh5Wk7Jr3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Steif C;Hinz HJ;Cesareni G,Proteins (1995) Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies. PMID:8539253
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
1B6Q 1999-01-16T00:00:00+0000 1.8 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN
1F4M 2000-06-08T00:00:00+0000 2.25 P3(2) CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
1F4N 2000-06-08T00:00:00+0000 1.9 C2 CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
1GMG 2001-09-13T00:00:00+0000 1.9 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN, MONOCLINIC FORM
1GTO 1996-04-23T00:00:00+0000 1.82 HIGH RESOLUTION STRUCTURE OF A HYPERSTABLE HELICAL BUNDLE PROTEIN MUTANT
1NKD 1997-09-23T00:00:00+0000 1.09 ATOMIC RESOLUTION (1.07 ANGSTROMS) STRUCTURE OF THE ROP MUTANT <2AA>
1QX8 2003-09-04T00:00:00+0000 2.02 Crystal structure of a five-residue deletion mutant of the Rop protein
1ROP 1991-04-02T00:00:00+0000 1.7 STRUCTURE OF THE COL*E1 ROP PROTEIN AT 1.7 ANGSTROMS RESOLUTION
1RPO 1994-08-25T00:00:00+0000 1.4 RESTORED HEPTAD PATTERN CONTINUITY DOES NOT ALTER THE FOLDING OF A 4-ALPHA-HELICAL BUNDLE
1RPR 1991-10-09T00:00:00+0000 0 THE STRUCTURE OF COLE1 ROP IN SOLUTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Regulatory protein rop P03051 ROP_ECOLX