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
4P61 2014-08-27 1.34 CHICKEN TRIOSEPHOSPHATE ISOMERASE WITH LOOP6 MUTATIONS, V167P AND W168E.
4UNL 2015-02-04 1.5 Crystal structure of a single mutant (N71D) of triosephosphate isomerase from human
1R2R 2003-12-23 1.5 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
4OWG 2014-03-05 1.55 Crystal structure of rabbit muscle triosephosphate isomerase-PEP complex
4POC 2015-01-14 1.6 Structure of Triosephosphate Isomerase Wild Type human enzyme.
2JK2 2008-07-01 1.7 STRUCTURAL BASIS OF HUMAN TRIOSEPHOSPHATE ISOMERASE DEFICIENCY. CRYSTAL STRUCTURE OF THE WILD TYPE ENZYME.
4ZVJ 2016-03-09 1.7 Structure of human triose phosphate isomerase K13M
1SW0 2004-08-24 1.71 Triosephosphate isomerase from Gallus gallus, loop 6 hinge mutant K174L, T175W
1TPH 1994-04-30 1.8 1.8 ANGSTROMS CRYSTAL STRUCTURE OF WILD TYPE CHICKEN TRIOSEPHOSPHATE ISOMERASE-PHOSPHOGLYCOLOHYDROXAMATE COMPLEX
2VOM 2008-06-17 1.85 Structural basis of human triosephosphate isomerase deficiency. Mutation E104D and correlation to solvent perturbation.
1TPB 1995-02-14 1.9 OFFSET OF A CATALYTIC LESION BY A BOUND WATER SOLUBLE
1TPC 1995-02-14 1.9 OFFSET OF A CATALYTIC LESION BY A BOUND WATER SOLUBLE
1TPW 1995-04-20 1.9 TRIOSEPHOSPHATE ISOMERASE DRINKS WATER TO KEEP HEALTHY
1TPV 1995-04-20 1.9 S96P CHANGE IS A SECOND-SITE SUPPRESSOR FOR H95N SLUGGISH MUTANT TRIOSEPHOSPHATE ISOMERASE
1TPU 1995-04-20 1.9 S96P CHANGE IS A SECOND-SITE SUPPRESSOR FOR H95N SLUGGISH MUTANT TRIOSEPHOSPHATE ISOMERASE
4BR1 2013-06-12 1.9 Protease-induced heterodimer of human triosephosphate isomerase.
1KLU 2002-08-02 1.93 Crystal structure of HLA-DR1/TPI(23-37) complexed with staphylococcal enterotoxin C3 variant 3B2 (SEC3-3B2)
4POD 2015-01-14 1.99 Structure of Triosephosphate Isomerase I170V mutant human enzyme.
4UNK 2015-02-04 2.0 Crystal structure of human triosephosphate isomerase (mutant N15D)
1SW3 2004-08-24 2.03 Triosephosphate isomerase from Gallus gallus, loop 6 mutant T175V
6D43 2018-05-16 2.04 CHARACTERIZATION OF HUMAN TRIOSEPHOSPHATE ISOMERASE S-NITROSYLATION
1SPQ 2004-08-24 2.16 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
6NLH 2019-06-19 2.2 Structure of human triose phosphate isomerase R189A
1WYI 2005-04-12 2.2 human triosephosphate isomerase of new crystal form
1SW7 2004-08-24 2.22 Triosephosphate isomerase from Gallus gallus, loop 6 mutant K174N, T175S, A176S
1R2T 2003-12-23 2.25 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
6C2G 2018-03-21 2.3 Human triosephosphate isomerase mutant V231M
1KLG 2002-08-02 2.4 Crystal structure of HLA-DR1/TPI(23-37, Thr28-->Ile mutant) complexed with staphylococcal enterotoxin C3 variant 3B2 (SEC3-3B2)
1TIM 1976-10-15 2.5 STRUCTURE OF TRIOSE PHOSPHATE ISOMERASE FROM CHICKEN MUSCLE
8TIM 1999-02-16 2.5 TRIOSE PHOSPHATE ISOMERASE
4E41 2012-08-29 2.6 Structural basis for the recognition of mutant self by a tumor-specific, MHC class II-restricted T cell receptor G4
1SU5 2004-08-24 2.7 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
2IAN 2007-04-03 2.8 Structural basis for recognition of mutant self by a tumor-specific, MHC class II-restricted TCR
1HTI 1995-01-26 2.8 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN TRIOSEPHOSPHATE ISOMERASE AT 2.8 ANGSTROMS RESOLUTION. TRIOSEPHOSPHATE ISOMERASE RELATED HUMAN GENETIC DISORDERS AND COMPARISON WITH THE TRYPANOSOMAL ENZYME
2IAM 2007-04-03 2.8 Structural basis for recognition of mutant self by a tumor-specific, MHC class II-restricted TCR
1R2S 2003-12-23 2.85 CRYSTAL STRUCTURE OF RABBIT MUSCLE TRIOSEPHOSPHATE ISOMERASE
1SQ7 2004-08-24 2.85 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SSD 2004-08-24 2.9 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase
1SSG 2004-08-24 2.9 Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase

Relevant UniProtKB Entries

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