Thermodynamic Basis for the Increased Thermostability of CheY from the Hyperthermophile <i>Thermotoga maritima</i>

Deutschman, W. A.; Dahlquist, Frederick W.

doi:10.1021/bi010665t

Cited by 37 publications

(33 citation statements)

References 22 publications

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“…Of the five thermodynamically characterized thermophile-mesophile protein pairs, four thermophilic proteins have up-shifted and broadened stability curves compared with their mesophilic homologs (8,(22)(23)(24)(25). It is difficult to make conclusions based on this limited data set, but it is possible that a large group of enzymes and other proteins requiring a finely tuned energy landscapes use lower ⌬Cp°values to balance a high melting temperature with the thermodynamic stability needed for optimal activity.…”

Section: Construction and Purification Of Variant Rnases Hmentioning

confidence: 99%

Role of residual structure in the unfolded state of a thermophilic protein

Robic

Guzmán-Casado²,

Sanchez-Ruiz³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

120

126

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Ribonucleases H from the thermophilic bacterium Thermus thermophilus and the mesophile Escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (⌬Cp°). The lower ⌬Cp°of the thermophilic protein directly contributes to its higher thermal denaturation temperature (T m). We propose that this ⌬Cp°difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a high T m with the optimal thermodynamic stability for a protein's function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of thermophilic and mesophilic proteins.T hermophilic organisms thrive at temperatures where proteins from mesophilic organisms are often completely unfolded and nonfunctional. Understanding the mechanisms by which proteins function at such high temperatures will help to optimize and design thermostable functional proteins for a variety of biotechnological applications. To learn how proteins from thermophilic organisms (thermophilic proteins) function at such elevated temperatures, we need to understand what makes these proteins different from their mesophilic homologs. This difference clearly does not reside in the overall structure of the native conformation; structures of numerous pairs of homologous proteins show that the thermophilic and mesophilic proteins invariably adopt the same fold (1). Examining the differences between individual amino acids and their specific interactions has led to the conclusion that the rules are extremely complex (2, 3).Thermodynamic comparisons of thermophilic and mesophilic pairs of proteins can provide a rational framework for understanding the functional differences between thermophilic and mesophilic proteins. A protein's thermodynamic stability is defined as the difference between the free energies of the native and the unfolded states (⌬G unf ϭ G U Ϫ G N ). The manner in which protein stability depends on temperature is illustrated by the so-called ''protein stability curve,'' which is defined by the Gibbs-Helmholtz equation (4, 5) (for an example, see Fig. 4). The temperature at which the ⌬G unf°e quals zero is the thermal denaturation midpoint (T m ), and the curvature of the stability curve, determined under standard conditions, is given by the heat capacity change upon unfolding (⌬Cp°). There are several ways in which a protein stability curve can be altered to result in a larger T m . An increase in the number of enthalpic interactions will raise the curve and make the protein more stable at every temperature. Alternatively, a lowering of the ⌬Cp°produces a ''flatter'' curve, which results in a higher T m for the same stability maximum. The question then becomes, how do thermophilic proteins alter these protein stability curves and how are they encoded in the structure and sequence?Thermus thermophilus and Escherichia coli RNa...

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Section: Construction and Purification Of Variant Rnases Hmentioning

confidence: 99%

Role of residual structure in the unfolded state of a thermophilic protein

Robic

Guzmán-Casado²,

Sanchez-Ruiz³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

120

126

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“…Equilibrium denaturation studies have provided detailed knowledge of the structure, stabilization, and folding of small, monomeric proteins [13,14]. Thermodynamic comparison of mesophilic and thermophilic ribonuclease H revealed that the heat capacity of unfolding of the thermophilic enzyme is lower than that of its mesophilic counterpart, although the proteins have maximal stabilities at similar temperatures [13].…”

Section: Introductionmentioning

confidence: 99%

“…Thermodynamic comparison of mesophilic and thermophilic ribonuclease H revealed that the heat capacity of unfolding of the thermophilic enzyme is lower than that of its mesophilic counterpart, although the proteins have maximal stabilities at similar temperatures [13]. For two small single-domain CheY homologues from Thermotoga maritima and Bacillus subtilis, it was found that the enhanced thermostability of the former is a direct result of the increased enthalpy contribution at the temperature of zero entropy, T s , and the decreased heat capacity change upon unfolding [14]. In addition, the dimeric serine hydroxymethyltransferases from B. subtilis and Geobacillus stearothermophilus followed different unfolding paths despite having a high degree of sequence identity [15].…”

Section: Introductionmentioning

confidence: 99%

“…4B, the stability curve for TMAI was shifted to a much higher DG U at all temperatures as compared to GSAI and BHAI, indicating that enthalpic contributions to the stability of the hyperthermophilic protein are much greater than the effects of the reduced DC p and changes in DS. As a result, the height of the stability curve rises, resulting in an exceptionally stable protein at moderate temperatures as well as an increase in the temperature of thermal unfolding [14,31].…”

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confidence: 99%

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A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic l‐arabinose isomerases: The effects of divalent metal ions on protein stability at elevated temperatures

et al. 2005

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To gain insight into the structural stability of homologous homo-tetrameric L L-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DG U ) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn 2+ , those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (T m ) due to decreased heat capacity (DC p ). Moreover, the extent of difference in DC p between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DC p , may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.

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“…It is interesting to study the change in intemal dynamics under the influence of various perturbations. Temperature is one of the important factors, since it can provide a probe of many aspects such as protein stability, protein folding, and ligand binding [11][12][13][14], as well as ah experimental means for investigating the energy landscape [15]. The temperature dependence of iSN autorelaxation rates in the backbone have been studied for a number of proteins [13,14,[16][17][18][19][20][21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Temperature-dependent protein backbone dynamics from auto- and cross-correlated NMR relaxation rates

Vugmeyster

Bodenhausen

2005

Appl. Magn. Reson.

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The temperature dependence of nuclear magnetic resonance relaxation rates was investigated for the backbone of tSN/~3C labeled human ubiquitin in the temperature range of 20-50 ~ The ~SN autorelaxation rates give evidente that the potential energy functions for ~SN-HN bonds ate not quadratic, in agreement with results for other proteins. Cross-correlation rates arising from correlated fluctuations of two ~SN-HN dipole~iipole interactions involving successive residues were obtained by the method of Petupessy et al. (E Pelupessy, S. Ravindranathan, G. Bodenhausen: J. Biomol. NMR 25, 265-280, 2003). The results suggest the presente of slow internal motions at 50 ~

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Thermodynamic Basis for the Increased Thermostability of CheY from the Hyperthermophile Thermotoga maritima

Cited by 37 publications

References 22 publications

Role of residual structure in the unfolded state of a thermophilic protein

Role of residual structure in the unfolded state of a thermophilic protein

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic l‐arabinose isomerases: The effects of divalent metal ions on protein stability at elevated temperatures

Temperature-dependent protein backbone dynamics from auto- and cross-correlated NMR relaxation rates

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