Disruption of an ionic network leads to accelerated thermal denaturation of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima

Pappenberger, G.; Schurig, Hartmut; Jaenicke, Rainer

doi:10.1006/jmbi.1997.1421

Cited by 97 publications

(70 citation statements)

References 49 publications

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“…In this network, Arg20 is connected to three other residues by ion pairs or H bonds. The mutations Arg20Ala and Arg20Asn increased the enzyme denaturation rate at 100°C by a factor of 3.5 (270). In T. maritima indoleglycerol phosphate synthase, the stabilization provided by ion pair Arg241-Glu73 (between [␣/␤] 8 barrel helices ␣ 8 and ␣ 1 ) was also tested by SDM.…”

Section: Ion Pairsmentioning

confidence: 99%

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Vieille¹,

Zeikus

2001

Microbiol Mol Biol Rev

1,847

1,476

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Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described

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Section: Ion Pairsmentioning

confidence: 99%

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Vieille¹,

Zeikus

2001

Microbiol Mol Biol Rev

1,847

1,476

View full text Add to dashboard Cite

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“…We assumed that the soluble molecules of TyrRS(Á1) were in a dimeric state and that the¯uorescence intensity was proportional to the concentration of dimer in the conditions of the measurement, 25 C in water. We then applied the transition state theory to translate the rate constant of precipitation into a free energy of activation, ÁG { , which is a measure of the kinetic stability (Pappenberger et al, 1997). From the values of ÁG { , we calculated the variations ÁÁG { of free energy when going from the wild-type TyrRS(Á1) to each of its mutants ( Table 2).…”

Section: Unfolding By Thermal Treatmentmentioning

confidence: 99%

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

Park

Guez

Bedouelle

1999

Journal of Molecular Biology

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A dense cluster of eight residues was identi®ed at the crossing of two a-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Á1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Á1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Á1) and its mutant derivatives at 68.5 C were determined from experiments of irreversible thermal precipitation. They were in the order L105V < I52L < T51P < Wild Type 4 M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Á1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectro¯uorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A Ê away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wildtype TyrRS(Á1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the ®rst pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.

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“…Buried salt bridges and salt bridges at dimer interfaces as well as surface salt bridges have been reported to contribute to the increased stability of proteins (7-11). These findings appear to contradict other thermodynamic evidence which shows that surface ion pairs make little contribution to protein stability (12)(13)(14).It has been difficult to study hyperthermophilic proteins by thermodynamic analysis, since most of the known hyperthermophilic proteins do not unfold reversibly (15)(16)(17)(18)(19)(20)(21)(22)(23). We previously designed a Pyroccocus furiosus rubredoxin variant 1 to eliminate the iron-binding site using a computational design algorithm.…”

mentioning

confidence: 99%

Contribution of Surface Salt Bridges to Protein Stability^,

2000

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The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1.5 kcal mol -1 . The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.Proteins from thermophilic organisms offer good model systems with which to address the origins of thermostability. Several trends were found to be associated with increased thermal stability. These trends include increased packing density, increased core hydrophobicity, decreased length of surface loops, and improved secondary structure stabilization such as N-terminal and C-terminal helix capping (1).Comparison of many thermophilic proteins also indicates a high level of occurrence of surface electrostatic interactions relative to mesophilic homologues (1-6). Buried salt bridges and salt bridges at dimer interfaces as well as surface salt bridges have been reported to contribute to the increased stability of proteins (7-11). These findings appear to contradict other thermodynamic evidence which shows that surface ion pairs make little contribution to protein stability (12)(13)(14).It has been difficult to study hyperthermophilic proteins by thermodynamic analysis, since most of the known hyperthermophilic proteins do not unfold reversibly (15)(16)(17)(18)(19)(20)(21)(22)(23). We previously designed a Pyroccocus furiosus rubredoxin variant 1 to eliminate the iron-binding site using a computational design algorithm. The iron-binding site in wild-type rubredoxin is thought to be the cause of its irreversible unfolding (22). The mutations for PFRD-XC4 are C5L, C8T, C38A, and C41T. PFRD-XC4 is able to fold in the absence of iron, undergoes reversible denaturation, and has a melting temperature of 82°C (24). The variant provides an excellent opportunity for systematic exploration of the factors determining protein thermostability.The NMR solution structure of PFRD-XC4 reveals a fold similar to that of wild-type P. furiosus rubredoxin (PFRD). Given that PFRD-XC4 adopts a fold similar to that of PFRD and undergoes reversible unfolding, the importance of interactions that are responsible for the extreme stability of PFRD can now be addressed. Two interactions found in PFRD, but not in mesophilic rubredoxins, have been proposed to contribute to its hyperthermostability (5, 25). These are the salt bridges between the N-terminus and the side chain of Glu 14 and between the side chains of Lys 6 and Glu 49. Here we present the NMR solution structure of PFRD-XC4 and thermodynamic analysis of the two salt bridges by double mutant cycle...

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Disruption of an ionic network leads to accelerated thermal denaturation of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima

Cited by 97 publications

References 49 publications

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

Contribution of Surface Salt Bridges to Protein Stability^,

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Disruption of an ionic network leads to accelerated thermal denaturation of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima

Cited by 97 publications

References 49 publications

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

Contribution of Surface Salt Bridges to Protein Stability,

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Contribution of Surface Salt Bridges to Protein Stability^,