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Stabilization of Human Triosephosphate Isomerase by Improvement of the Stability of Individual α-Helices in Dimeric as well as Monomeric Forms of the Protein†

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Stabilization of Human Triosephosphate Isomerase by Improvement of the Stability of Individual α-Helices in Dimeric as well as Monomeric Forms of the Protein†
 
Creator Mainfroid, Véronique
Mande, Shekhar C
Hol, Wim G. J.
Martial, Joseph A.
Goraj, K
 
Subject QR Microbiology
 
Description Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting
the R/â-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, 2.5 kcal/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 R-helices of the protein. Five mutations were proposed, designed
to increase R-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 R-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.
 
Publisher ACS Publications
 
Date 1996
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/735/1/mande1996.1.pdf
Mainfroid, Véronique and Mande, Shekhar C and Hol, Wim G. J. and Martial, Joseph A. and Goraj, K (1996) Stabilization of Human Triosephosphate Isomerase by Improvement of the Stability of Individual α-Helices in Dimeric as well as Monomeric Forms of the Protein†. Biochemistry, 35 (13). pp. 4110-4117. ISSN 0006-2960
 
Relation http://dx.doi.org/10.1021/bi952692n
http://crdd.osdd.net/open/735/