Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein.


Abstract

Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting the alpha/beta-barrel fold. In a previous work, a monomeric mutant of hTIM was engineered in which Met14 and Arg98, two interface residues, were changed to glutamine. Analysis of equilibrium denaturation of this monomeric mutant, named M14Q/R98Q, revealed that its conformational stability, 2.5kcal/mol, is low as compared to the stability of dimeric hTIM (19.3 kcal/mol). The fact that this value is also lower than the conformational stabilities usually found for monomeric proteins suggests that the hTIM monomers are thermodynamically unstable. In the present work, we attempted to stabilize the M14Q/R98Q mutant by introducing stabilizing mutations in alpha-helices of the protein. Five mutations were proposed, designed to increase alpha-helix propensity by introducing alanines at solvent-exposed sites (Q179A, K193A), to introduce favorable interactions with helix dipoles (Q179D, S105D), or to reduce the conformational entropy of unfolding by introducing proline residues at the "N-cap" position of alpha-helices (A215P). Three replacements (Q179D, K193A, and A215P) were found to increase the stability of the native dimeric hTIM and the monomeric M14Q/R98Q. These results suggest that the monomeric hTIM mutant can be stabilized to a considerable extent by following well-established rules for protein stabilization. A comparison of the stabilizing effect performed by the mutations on the dimeric hTIM and the monomeric M14Q/R98Q allowed us to reinforce a model of equilibrium denaturation proposed for both proteins. Study holds ProTherm entries: 5017, 5018, 5019, 5020, 5021, 5022, 5023 Extra Details:

Submission Details

ID: mycNT3cD

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Mainfroid V;Mande SC;Hol WG;Martial JA;Goraj K,Biochemistry (1996) Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein. PMID:8672446
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
1R2R 2003-09-29T00:00:00+0000 1.5 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
1R2S 2003-09-29T00:00:00+0000 2.85 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
1R2T 2003-09-29T00:00:00+0000 2.25 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
4OWG 2014-02-01T00:00:00+0000 1.55 Crystal structure of rabbit muscle triosephosphate isomerase-PEP complex
1SPQ 2004-03-17T00:00:00+0000 2.16 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SQ7 2004-03-18T00:00:00+0000 2.85 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SSD 2004-03-24T00:00:00+0000 2.9 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SSG 2004-03-24T00:00:00+0000 2.9 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SU5 2004-03-26T00:00:00+0000 2.7 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SW0 2004-03-30T00:00:00+0000 1.71 Triosephosphate isomerase from Gallus gallus, loop 6 hinge mutant K174L, T175W

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Triosephosphate isomerase P60174 TPIS_HUMAN
98.4 Triosephosphate isomerase P00939 TPIS_RABIT
96.0 Triosephosphate isomerase P17751 TPIS_MOUSE
100.0 Triosephosphate isomerase P60175 TPIS_PANTR
99.2 Triosephosphate isomerase Q60HC9 TPIS_MACFA
99.2 Triosephosphate isomerase P15426 TPIS_MACMU
99.2 Triosephosphate isomerase Q2QD07 TPIS_GORGO
99.6 Triosephosphate isomerase Q5R928 TPIS_PONAB
98.4 Triosephosphate isomerase P54714 TPIS_CANLF
97.6 Triosephosphate isomerase Q5E956 TPIS_BOVIN
94.4 Triosephosphate isomerase P48500 TPIS_RAT
93.5 Triosephosphate isomerase Q29371 TPIS_PIG
90.2 Triosephosphate isomerase P00940 TPIS_CHICK