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See detailStabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein
Mainfroid, Véronique; Mande, Shekhar C; Hol, Wim G J et al

in Biochemistry (1996), 35(13), 4110-7

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 ... [more ▼]

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. [less ▲]

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See detailThree hTIM mutants that provide new insights on why TIM is a dimer
Mainfroid, Véronique; Terpstra, Peter; Beauregard, Marc et al

in Journal of Molecular Biology (1996), 257(2), 441-56

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in ... [more ▼]

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studies, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less active than unmutated hTIM. Moreover, its specific activity is concentration dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of the wild-type and mutant hTIMs, unfolding of all three enzymes was monitored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. The conformational stability of hTIM, as estimated according to this model, is 19.3 (+/-0.4) kcal/mol. The M14Q and R98Q replacements significantly reduce enzyme stability, since the free energies of unfolding are 13.8 and 13.5 (+/- 0.3) kcal/mol respectively, for the mutants, A third mutant, in which the M14Q and R98Q replacements are cumulated, behaves like a monomer. The stability of this mutant is not concentration-dependent, and the unfolding reaction is assigned to a transition from a folded monomer to an unfolded monomer. The conformational stability of this double mutant is estimated 2.5 (+/-0.1) kcal/mol. All these data combined suggest that TIM monomers are thermodynamically unstable. This might explain why TIM occurs only as a dimer. [less ▲]

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See detailSecond-generation octarellins: two new de novo (beta/alpha)8 polypeptides designed for investigating the influence of beta-residue packing on the alpha/beta-barrel structure stability
Houbrechts, Annick ULg; Moreau, Benoît; Abagyan, Ruben et al

in Protein Engineering (1995), 8(3), 249-59

The sequence of octarellin I, the first de novo (beta/alpha)8 polypeptide, was revised according to several criteria, among others the symmetry of the sequence, beta-residue volume and hydrophobicity, and ... [more ▼]

The sequence of octarellin I, the first de novo (beta/alpha)8 polypeptide, was revised according to several criteria, among others the symmetry of the sequence, beta-residue volume and hydrophobicity, and charge distribution. These considerations and the overall conclusions drawn from the first design led to two new sequences, corresponding to octarellins II and III. Octarellin II retains perfect 8-fold symmetry. Octarellin III has the same sequence as octarellin II, except for the beta-strands which exhibit a 4-fold symmetry. The two proteins were produced in Escherichia coli. Infrared and CD spectral analyses of octarellins II and III reveal a high secondary structure content. Non-denaturing gel electrophoresis, molecular sieve chromatography and analytical ultracentrifugation suggest that both of these second-generation artificial polypeptides exist as a mixture of a monomer and a dimer form. Octarellins II and III are at least 10 times more soluble than octarellin I. Urea-induced unfolding followed by fluorescence emission suggests that the tryptophan residues, designed to be buried in the (beta/alpha)8, are indeed packed in the hydrophobic core of both proteins. However, octarellin III displays a higher stability towards urea denaturation, indicating that introducing 4-fold symmetry into the beta-barrel might be important for stability of the overall folding. [less ▲]

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See detailModular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM
Kishan, Radha; Zeelen, Ph. Johan; Noble, Martin E.M. et al

in Protein Engineering (1994), 7(8), 945-51

The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha ... [more ▼]

The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E. coli TIM with the equivalent unit of chicken TIM. This replacement involves 10 sequence changes. One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities. The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8). From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM. [less ▲]

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See detailCloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria. Structural comparison of the predicted protein sequences
Rentier-Delrue, Françoise ULg; Mande, Shekhar C; Moyens, Sylvianne et al

in Journal of Molecular Biology (1993), 229(1), 85-93

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments ... [more ▼]

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues. [less ▲]

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See detailReplacing the (beta alpha)-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme
Mainfroid, Véronique; Goraj, Karine; Rentier-Delrue, Françoise ULg et al

in Protein Engineering (1993), 6(8), 893-900

In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein ... [more ▼]

In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein, assuming that the pseudosymmetrical beta-barrel can be divided into eight successive loop/beta-strand/loop/alpha-helix motifs. We replaced the eighth (beta alpha)-unit of E.coli TIM with the corresponding chicken (beta alpha)-unit. The substitution, involving the replacement of 10 of the 23 residues of this (beta alpha)-unit, was evaluated first by modelling, then experimentally. Modelling by homology suggests how the amino acid replacements might be accommodated in the hybrid E.coli/chicken TIM (ETIM8CHI). Both natural and hybrid recombinant TIMs, overproduced in E.coli, were purified to homogeneity and characterized as to their stability and kinetics. Our kinetic studies show that the modification performed here leads to an active enzyme. The stability studies indicate that the stability of ETIM8CHI is comparable to that of the wild type TIM. [less ▲]

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See detailSpectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the alpha/beta-barrel packing)
Beauregard, Marc; Goraj, Karine; Goffin, Vincent et al

in Protein Engineering (1991), 4(7), 745-9

We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein ... [more ▼]

We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested. [less ▲]

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See detailSynthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the alpha/beta-barrel proteins
Goraj, Karine; Renard, André; Martial, Joseph ULg

in Protein Engineering (1990), 3(4), 259-66

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt ... [more ▼]

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern. A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles. The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units. Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins. Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions. [less ▲]

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