2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

Hennig, Michael; Darimont, Beatrice; Sterner, Reinhard; Kirschner, Kasper; Jansonius, Johan N.

doi:10.1016/s0969-2126(01)00267-2

Cited by 240 publications

(254 citation statements)

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“…The charged residues are likely to favor ion pairs, hydration interactions, and hydrogen bonds. Ion pairs have been proposed to play a key role in the maintenance of enzyme stability at high temperature (41,42). The number of intrasubunit ion pairs in Mj MalDH is increased significantly (50%) compared with its mesophilic counterparts (TABLE TWO), confirming the proposed stabilizing effect of ion pairs.…”

Section: Resultsmentioning

confidence: 78%

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Tehei

Madern

Franzetti

et al. 2005

Journal of Biological Chemistry

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To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.Hyperthermophilic organisms grow at temperatures above 80°C. Proteins from these organisms are themselves optimally active between 60 and 125°C and serve as paradigms for the characterization of factors responsible for protein fold stability and flexibility. Hyperthermophilic enzymes have also attracted considerable attention because of a range of biotechnological applications (1, 2).Sequence comparison studies and structural analyses carried out on hyperthermophilic proteins and their mesophilic homologues have shown that thermal stability is associated with multiple factors, including an increase in hydrogen bonding, complex salt bridge formation, and helix stabilization by acidic residues. The commonly accepted hypothesis is that increased thermal stability is due to enhanced conformational rigidity of the molecular structure (3). Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3, 4). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity.Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4, 5), whereas others, on ␣-amylase (6) and on rubredoxin (7,8), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects. Relations between flexibility and stability are, therefore, complex. Atomic fluctuations only were measured in these experiments and interpreted in terms of flexibility.It is important to point out that reduced structural "flexibility" does not necessarily imply a more "rigid" structure. Atoms are maintained in a structure by forces that link them to their neighbors. In terms of a force field, the width of the potential well in which an atom moves is a measure of its flexibility in terms of a root mean square fluctuation amplitude ͑ ͌ Ͻu 2 Ͼ), whereas the detailed ...

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Section: Resultsmentioning

confidence: 78%

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Tehei

Madern

Franzetti

et al. 2005

Journal of Biological Chemistry

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“…The disulfide bond is accessible (ASA values of T3 and R189 are 54 and 68 Å 2 , respectively) and fortuitously mimics one of the extra salt bridges in both sIGPS [7] and tIGPS [3], which are missing in eIGPS [1]. However, the replacement R189C in eIGPS disrupts the parental shortrange salt bridge of R189 to E169 on helix a5 .…”

Section: Design Of Disulfide Bondsmentioning

confidence: 99%

“…In contrast, ePRAI activity decays at 60°C with a half-life of 100 min (R. Sterner, Institut fu¨r Biochemie, Universita¨t zu Kö ln, Germany, personal communication). The eIGPS domain, in turn, is also more labile than eIGPS in the native bifunctional protein [4,5,6].In contrast to eIGPS [1], the IGP synthases from the hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and Thermotoga maritima (tIGPS [3]), are thermostable, monofunctional monomers. The comparison of the three high resolution crystal structures suggests that an increased number of salt bridges over that in eIGPS decreases the rates of irreversible thermal inactivation of both sIGPS and tIGPS.…”

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confidence: 99%

Stabilization of a (βα)₈‐barrel protein by an engineered disulfide bridge

Ivens

Mayans²,

Szadkowski

et al. 2002

European Journal of Biochemistry

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The aim of this study was to increase the stability of the thermolabile (ba) 8 -barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges. For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines. The variant linking residues 3 and 189 fastens the N-terminus to the (ba) 8 -barrel. The rate of thermal inactivation at 50°C of this variant with a closed disulfide bridge is 65-fold slower than that of the reference dithiol form, but only 13-fold slower than that of the parental protein. The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified. To confirm these data, we have solved the X-ray structure to 2.1-Å resolution. The second variant was designed to crosslink the terminal modules ba1 and ba8. However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein.Keywords: indoleglycerol phosphate synthase; (b/a) 8 -barrel proteins; stabilizing disulfide bonds; protein engineering.Indoleglycerol phosphate synthase (IGPS) is a (ba) 8 -barrel protein with an N-terminal extension of 48 residues. In Escherichia coli, IGPS (eIGPS) is the N-terminal domain of a monomeric, bifunctional enzyme, where the C-terminal domain is phosphoribosyl anthranilate isomerase (ePRAI), folded into another (ba) 8 -barrel [1]. The catalytic efficiencies of the engineered separated domains are virtually identical to those in the bifunctional enzyme [2]. eIGPS is, however, more labile than ePRAI. The catalytic activity of eIGPS decays at 55°C with a half-life of 0.5 min [3]. In contrast, ePRAI activity decays at 60°C with a half-life of 100 min (R. Sterner, Institut fu¨r Biochemie, Universita¨t zu Kö ln, Germany, personal communication). The eIGPS domain, in turn, is also more labile than eIGPS in the native bifunctional protein [4,5,6].In contrast to eIGPS [1], the IGP synthases from the hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and Thermotoga maritima (tIGPS [3]), are thermostable, monofunctional monomers. The comparison of the three high resolution crystal structures suggests that an increased number of salt bridges over that in eIGPS decreases the rates of irreversible thermal inactivation of both sIGPS and tIGPS. In support of this proposal, mutational disruption of salt bridge that crosslinks its terminal ba1 and ba8 modules, significantly destabilized the variants by comparison to the parental enzyme [3], in support of analogous findings reported previously [8].The aim of this work was to stabilize the labile eIGPS domain by introducing new disulfide bonds rather than new salt bridges. Disulfide bonds can stabilize p...

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“…Therefore, researchers in both scientific and industrial fields are interested in the factors responsible for protein thermostability. Various means for control of the thermostability have been studied and these include amino acid charge [1,2], electrostatic interactions [3][4][5][6][7][8], aromatic interactions [9], compactness [10], deamidation [11], and so on.…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of protein thermostability: Differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins

Yokota

Satou

Ohki³

2006

Science and Technology of Advanced Materials

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In order to investigate the factors responsible for protein thermostability, we performed a comparative analysis. For this study, we prepared a new dataset composed of 47 homologous pairs of thermophilic and mesophilic proteins. It is the largest comparative study dataset ever presented. The frequency and substitution preference of each amino acid type in the dataset were analyzed. Two kinds of residual structural states were considered, i.e. surface (solvent-exposed) and core (buried) regions. On the surface of thermophilic proteins, higher frequencies were observed for Arg, Glu, and Tyr. Analysis of substitution preference also suggests that these often appear by replacement of other amino acid types. The results indicate that Arg, Glu, and Tyr are suitable for location on the surface of thermophilic proteins. On the other hand, at the core of thermophilic proteins, Ala is often appeared. In addition, our t-test analysis provides the first quantitative information about trends in the frequencies and substitution preferences for Cys, Gln, Met, and Ser. The results indicate that Gln and Met on the surface and Cys and Ser in the core are disadvantageous for protein thermostability. q

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2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

Abstract: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Cited by 240 publications

References 50 publications

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Stabilization of a (βα)₈‐barrel protein by an engineered disulfide bridge

Comparative analysis of protein thermostability: Differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins

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2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

Abstract: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Cited by 240 publications

References 50 publications

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Stabilization of a (βα)8‐barrel protein by an engineered disulfide bridge

Comparative analysis of protein thermostability: Differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins

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Stabilization of a (βα)₈‐barrel protein by an engineered disulfide bridge