The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges 1 1Edited by R. Huber

Merz, Astrid; Knöchel, Thorsten; Jansonius, Johan N.; Kirschner, Kasper

doi:10.1006/jmbi.1999.2709

Cited by 69 publications

(67 citation statements)

References 45 publications

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supporting

confidence: 88%

“…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 proteins undergoing reversible unfolding by decreasing the main chain entropy of their unfolded states [9][10][11].…”

supporting

confidence: 87%

“…Data were recorded on a MAR-CCD detector in three resolution sweeps to a maximum resolution of 2.1 Å . The crystals belong to the space group P6 3 Only reflections corresponding to the subcell (k ¼ h ± 3n) show significant intensities, with reflections from the larger cell being remarkably weaker. This crystallographic problem also affected monomeric, wild-type eIGPS and has been described previously [25].…”

Section: X-ray Structure Solutionmentioning

confidence: 99%

“…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 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.…”

mentioning

confidence: 99%

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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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supporting

confidence: 88%

supporting

confidence: 87%

Section: X-ray Structure Solutionmentioning

confidence: 99%

Section: Design Of Disulfide Bondsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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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Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

et al. 2001

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Homeodomains are a class of helix–turn–helix DNA‐binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three α‐helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation–π interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild‐type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation–π interactions that connect secondary structures. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 145–159, 2001

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Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

Sehgal

Tompson

Cavanagh

et al. 2002

Biotech & Bioengineering

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The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.

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The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges 1 1Edited by R. Huber

Cited by 69 publications

References 45 publications

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

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

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

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The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges 1 1Edited by R. Huber

Cited by 69 publications

References 45 publications

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

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

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

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Product

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

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