Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization.


Abstract

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water. Study holds ProTherm entries: 319, 320, 321, 322, 323, 324, 2195 Extra Details: human lysozyme; refolding kinetics; proline mutations;,protein stability; energetics; folding pathway

Submission Details

ID: jX8vSQBA4

Submitter: Connie Wang

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

Version: 1

Publication Details
Herning T;Yutani K;Taniyama Y;Kikuchi M,Biochemistry (1991) Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization. PMID:1911779
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
133L 1993-06-01T00:00:00+0000 1.77 ROLE OF ARG 115 IN THE CATALYTIC ACTION OF HUMAN LYSOZYME. X-RAY STRUCTURE OF HIS 115 AND GLU 115 MUTANTS
134L 1993-06-01T00:00:00+0000 1.77 ROLE OF ARG 115 IN THE CATALYTIC ACTION OF HUMAN LYSOZYME. X-RAY STRUCTURE OF HIS 115 AND GLU 115 MUTANTS
1B5U 1999-01-11T00:00:00+0000 1.8 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANT
1B5V 1999-01-11T00:00:00+0000 2.17 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5W 1999-01-11T00:00:00+0000 2.17 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5X 1999-01-11T00:00:00+0000 2.0 Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and x-ray analysis of six ser->ala mutants
1B5Y 1999-01-11T00:00:00+0000 2.2 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B5Z 1999-01-11T00:00:00+0000 2.2 CONTRIBUTION OF HYDROGEN BONDS TO THE CONFORMATIONAL STABILITY OF HUMAN LYSOZYME: CALORIMETRY AND X-RAY ANALYSIS OF SIX SER->ALA MUTANTS
1B7L 1999-01-24T00:00:00+0000 1.8 VERIFICATION OF SPMP USING MUTANT HUMAN LYSOZYMES
1B7M 1999-01-24T00:00:00+0000 2.2 VERIFICATION OF SPMP USING MUTANT HUMAN LYSOZYMES

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lysozyme C P79179 LYSC_GORGO
100.0 Lysozyme C P61626 LYSC_HUMAN
100.0 Lysozyme C P61627 LYSC_PANPA
100.0 Lysozyme C P61628 LYSC_PANTR
100.0 Lysozyme C P79239 LYSC_PONPY
97.7 Lysozyme C P79180 LYSC_HYLLA
90.8 Lysozyme C P61633 LYSC_CHLAE
90.8 Lysozyme C P61634 LYSC_ERYPA