Serial increase in the thermal stability of 3‐isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution

Akanuma, Satoshi; Yamagishi, Akihiko; Tanaka, Nobuo; Oshima, Tairo

doi:10.1002/pro.5560070319

Cited by 76 publications

(46 citation statements)

References 37 publications

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“…Too much stability may also decrease activity, as proteins "breathe" and require mechanical flexibility for catalysis (James & Tawfik, 2003). Such a tradeoff between stability and activity has been demonstrated in in vitro studies on the evolution of thermostability (Akanuma et al, 1998) and drug resistance (Wang et al, 2002) and may well be a more general phenomenon (DePristo et al, 2005). Given that the effect of amino acid substitutions on G is in the order of 0.5 to 5 kcal/mol, a significant fraction of missense mutations would be expected to affect enzyme function (DePristo et al, 2005).…”

Section: Most Missense Mutations Affect Protein Functionmentioning

confidence: 99%

Genetic Constraints on Protein Evolution

Camps

Herman

Loh

et al. 2007

Critical Reviews in Biochemistry and Molecular Biology

123

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Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.

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Section: Most Missense Mutations Affect Protein Functionmentioning

confidence: 99%

Genetic Constraints on Protein Evolution

Camps

Herman

Loh

et al. 2007

Critical Reviews in Biochemistry and Molecular Biology

123

View full text Add to dashboard Cite

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“…Most attempts at discovering the origin of their stability have involved comparative thermodynamic and/or amino acid sequence analyses of homologous proteins from organisms living at different temperatures [1,2,[5][6][7][8][9]. Thus it is generally accepted that to arrive at a complete understanding of the thermal adaptation strategies of these proteins it is necessary to obtain and compare the unfolding thermodynamic functions of mutants and other family members.…”

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confidence: 99%

Thermodynamic analysis of the unfolding and stability of the dimeric DNA‐binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant

Ruiz-Sanz

Filimonov

Christodoulou

et al. 2004

European Journal of Biochemistry

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We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 lM of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJAEK ) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJAEmol )1 at 25°C for DDG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.Keywords: differential scanning microcalorimetry; hyperthermophilic HU protein; polar interactions; thermal stability; unfolding heat capacity.Proteins from thermophilic and hyperthermophilic microorganisms are of major interest to industrial biotechnology because they are usually more stable at high temperatures than their analogues from mesophilic organisms whilst they retain the folding patterns of their protein family [1][2][3][4]. Most attempts at discovering the origin of their stability have involved comparative thermodynamic and/or amino acid sequence analyses of homologous proteins from organisms living at different temperatures [1,2,[5][6][7][8][9]. Thus it is generally accepted that to arrive at a complete understanding of the thermal adaptation strategies of these proteins it is necessary to obtain and compare the unfolding thermodynamic functions of mutants and other family members.HU is a small histone-like bacterial protein that binds to DNA. It is abundant in all prokaryotes and its sequence is quite similar in a considerable number of species [10]. It is essential in the assembly of supramolecular nucleoprotein complexes and is also involved in a variety of DNA metabolic events, such as replication, transcription and transposition [11,12]. Its ability to repair DNA [13,14] and to prevent DNA duplex melting [7] has also been described.HU proteins from several species of bacillus growing in environments of different temperatures have already been isolated and st...

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“…In general, mutations affecting the thermostability of proteins show cumulative effects. 4,26) To obtain additional thermostabilized mutants, we introduced the mutations identified in the first screening into hyg4 and then tested them for thermostability. As expected, a small but distinct increase in thermostability was observed.…”

Section: Resultsmentioning

confidence: 99%

“…It is also easy to transform. Several successful studies have been done by this host-vector system, such as HTK for the kanamycin (Km) resistance gene from Staphylococcus aureus, 2) HTS for the bleomycin-resistance gene from Streptomyces hindustanus, 3) and the leuB genes from Bacillus subtilis 4) and Saccharomyces cerevisiae.…”

mentioning

confidence: 99%