Applications of NMR to Thermostable Proteins

Alberti, E.; Consonni, Roberto; Zetta, Lucia

doi:10.1016/s0066-4103(03)50003-5

Cited by 3 publications

(4 citation statements)

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“…Understanding the mechanism of protein stability under poly-extreme conditions such as high temperatures, a wide range of pH and resistance to degradation by proteases is a great challenge. Many studies have indicated that there is no single and unique structural requirement for making a protein stable under a variety of extreme conditions; several factors such as increased hydrophobic and aromatic contacts, electrostatic interactions and side chain packing [1] appear to play crucial roles in protein stability. However, the mechanism by which proteins attain the stability to function under poly-extreme conditions remains elusive.…”

Section: Introductionmentioning

confidence: 99%

The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

et al. 2008

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BackgroundUnderstanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature.MethodologyWe investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX.ConclusionsA single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

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Section: Introductionmentioning

confidence: 99%

The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

et al. 2008

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show abstract

“…Understanding the mechanism of protein stability under polyextreme conditions such as high temperatures, a wide range of pH and resistance to degradation by proteases is a great challenge. Many studies have indicated that there is no single and unique structural requirement for making a protein stable under a variety of extreme conditions; several factors such as increased hydrophobic and aromatic contacts, electrostatic interactions and side chain packing [1] appear to play crucial roles in protein stability. However, the mechanism by which proteins attain the stability to function under poly-extreme conditions remains elusive.…”

Section: Introductionmentioning

confidence: 99%

Correction: The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

Bhardwaj¹,

Leelavathi²,

Mazumdar-Leighton³

et al. 2009

PLoS ONE

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Background: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. Methodology:We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. Conclusions:A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo-and alkali-stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali-and SDS-sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

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“…The interest for thermophilic proteins has increased during the last years for several reasons: first of all they can open a wide field of microbiological and technical industrial applications and processes, furthermore, they provide excellent models for understanding the molecular mechanisms underlying protein folding and stabilization, and for describing their functional properties. The stability of proteins from organisms living under extreme conditions is a long standing issue and different authors attributed several factors affecting their greater structural stability with respect to the mesophilic counterpart 1. It has been pointed out that there is not a unique structural requirement for making a protein thermostable.…”

Section: Introductionmentioning

confidence: 99%

“…It has been pointed out that there is not a unique structural requirement for making a protein thermostable. Several factors have been found to play a relevant role in the protein stability, such as increased hydrophobic and aromatic contacts, optimization of charge–charge interactions, and side chain packing1; however, the mechanisms by which these proteins attain thermostability remain unclear.…”

Section: Introductionmentioning

confidence: 99%

Structural determinants responsible for the thermostability of Sso7d and its single point mutants

et al. 2007

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Sso7d is a small protein especially attractive as a model for characterizing by NMR, the structural determinants responsible for thermal stability. With this aim, structural parameters of the Sso7d protein have been compared with those of single point mutants, in particular with K12L, more stable (T m around 1008C) and F31A, less stable (T m 618C) than the wild-type. In addition, a mutant lacking eight residues of the C-terminal helix (T m 508C) has been studied to investigate the role of the helix in structure stabilization. Melting temperatures, DDG's of unfolding, cavities, electrostatic interactions, solvent accessible areas, hydrophobic cores, and aromatic cluster geometries have been analyzed in order to gain insights into the key structural factors determining the thermostability differences. Our data suggest that absence of cavities and a larger salt bridge network represent the major determinants contributing to the enhanced stability of K12L relative to the Sso7d protein and its mutant F31A.Introduction. The interest for thermophilic proteins has increased during the last years for several reasons: first of all they can open a wide field of microbiological and technical industrial applications and processes, furthermore, they provide excellent models for understanding the molecular mechanisms underlying protein folding and stabilization, and for describing their functional properties. The stability of proteins from organisms living under extreme conditions is a long standing issue and different authors attributed several factors affecting their greater structural stability with respect to the mesophilic counterpart. 1 It has been pointed out that there is not a unique structural requirement for making a protein thermostable. Several factors have been found to play a relevant role in the protein stability, such as increased hydrophobic and aromatic contacts, optimization of charge-charge interactions, and side chain packing 1 ; however, the mechanisms by which these proteins attain thermostability remain unclear.In the last years we have studied the recombinant form of Sso7d, initially isolated from the thermoacidophilic archeaobacterium Sulfolobus solfataricus living in volcanic hot springs at 878C and displaying a pressure resistance up to 14 kbar. 2 The protein is composed of 62

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Applications of NMR to Thermostable Proteins

Cited by 3 publications

References 356 publications

The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

Correction: The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

Structural determinants responsible for the thermostability of Sso7d and its single point mutants

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