Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Km, Perry; Jj, Onuffer; Ms, Gittelman; Barmat, L; Cr, Matthews

doi:10.1021/bi00445a061

Cited by 66 publications

(44 citation statements)

References 27 publications

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“…Asp134 is located in the center of the aV helix, which includes residues 127-141, suggesting that this position is independent of the specific preferences of amino acid residues at the N-termini and C-termini of the a helix [53, 541. Asp134 is partially exposed to the solvent, with an accessible surface area of 0.5 nm2 relative to 1.32 nm2 for the extended chain, and has relatively poor interactions with other residues, except for the salt bridge with Arg138, which does not seem to contribute greatly to the protein stability. This may be the reason why a relatively good correlation was observed between the stabilities of the mutant proteins and the rank [30,[48][49][50].…”

Section: Protein Stabilitymentioning

confidence: 99%

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

Haruki

Noguchi

Nakai

et al. 1994

European Journal of Biochemistry

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The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the nV helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. cali strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins rD134HIRNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp1 34 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the nV helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its 61 position impacts catalysis.Ribonuclease H (RNase H), which degrades only the RNA strand of an RNA . DNA hybrid in the presence of divalent cations, such as Mg" or Mn", is widely found in various organisms [ 1 -31. Eschericlziu coli RNase HI, which had been designated as RNase H until a second RNase H (RNase HII) was isolated from E. coli 141, is composed of a single polypeptide chain with 155 amino acid residues [S] and has a PI value of 9.0 [6]. The similarities in the primary structures of this enzyme, reverse transcriptase RNase H 17, 81, Thermus thermophilus RNase H [91, and yeast RNase H [lo] strongly suggest that these enzymes are evolutionally related. In fact, the three-dimensional structure of E. coli RNase HI [ll-131 is similar to that of the reverse-transcriptase RNase H from human immunodeficiency virus-1 (HIV-1) [14-161. Therefore, it has been anticipated that detailed analyses of the structure/function relationships of E. Correspondence to S . Kanaya, Protein Engineering ResearchFax: +81 6 872 8210. Abbreviutions. GdnHCI, guanidine hydrochloride; HIV-1, human immunodeficiency virus-1 ; PCR, polymerase chain reaction; t,,*, temperature at which an enzyme loses 50% of its activity; P,, conformational parameter for u-helical residues in proteins ; RNase, ribonuclease; [DI 34XIRNase H, mutant ribonuclease H in which aspartic acid at position 134 is replaced by a specified amino acid.Institute,

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Section: Protein Stabilitymentioning

confidence: 99%

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

Haruki

Noguchi

Nakai

et al. 1994

European Journal of Biochemistry

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“…25 Other groups reached similar conclusions based on m values. [26][27][28][29][30][31] Myers et al 32 showed that the GdnHCl and urea m values correlate with the amount of protein surface area newly exposed to solvent upon unfolding, as does the heat capacity change. Several groups have used different approaches and model compound data to account for this observation: Schellman, 33 Timasheff, 34 Record, 35 and Bolen.…”

Section: Introductionmentioning

confidence: 99%

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

Pace

Huyghues-Despointes

et al. 2010

Protein Science

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The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge-charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317-15323).

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“…The answer to this question has immediate importance for the biotechnological industry, which is interested in improving the thermostability of enzymes. The efforts in this direction have concentrated on repacking the hydrophobic cores, engineering disulfide bridges, adding extra hydrogen bonds or salt bridges, and improving secondary structure propensities or side chain helix dipole interactions (1)(2)(3)(4)(5)(6)(7)(8). All these methods can be characterized as optimizing the local short-range interactions.…”

mentioning

confidence: 99%

Engineering a Thermostable Protein via Optimization of Charge−Charge Interactions on the Protein Surface

et al. 1999

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A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wildtype protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.How to stabilize protein structure? This question is not just of scholastic interest. The answer to this question has immediate importance for the biotechnological industry, which is interested in improving the thermostability of enzymes. The efforts in this direction have concentrated on repacking the hydrophobic cores, engineering disulfide bridges, adding extra hydrogen bonds or salt bridges, and improving secondary structure propensities or side chain helix dipole interactions (1-8). All these methods can be characterized as optimizing the local short-range interactions. Such an approach is well justified because it is becoming more and more clear that the protein folding is a hierarchical process and thus is mostly driven by local interactions (9, 10). However, this does not mean that the final native state of the protein is stabilized exclusively by the short-range interactions (11,12). Long-range interactions such as chargecharge interactions will also contribute to the stability of proteins. To address this issue, we developed a simple approach [the "TK-BK procedure" (13)] that allows us, nevertheless, to determine the regions of the protein surface where redesign of the charge-charge interactions is likely to lead to stability enhancement. In the TK-BK procedure (13), the interaction energies between unit positive charges placed in the protonation sites of ionizable groups are estimated using the solvent-accessibility-corrected Tanford-Kirkwood model (14), and subsequently, the pairwise charge-charge interaction energies are calculated as averages over the protonation states of the native protein on the basis of the BashfordKarplus reduced-set-of-sites approximation (15). This approach is indeed a simple one (charge-charge intera...

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Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase

Cited by 66 publications

References 27 publications

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

Engineering a Thermostable Protein via Optimization of Charge−Charge Interactions on the Protein Surface

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