Backbone Dynamics of the C-Terminal Domain of <i>Escherichia coli</i> Topoisomerase I in the Absence and Presence of Single-Stranded DNA

Yu, Liping; Zhu, Chang-Xi; Tse‐Dinh, Yuk‐Ching; Fesik, Stephen W.

doi:10.1021/bi960507f

Cited by 78 publications

(89 citation statements)

References 22 publications

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“…Thus, separation of the backbone entropy term from other terms is only possible through the use of methods, such as 15 N NMR relaxation, that are selectively sensitive to backbone motions. Recent studies have indicated that increases in protein conformational entropy upon ligand binding may help to stabilize protein-ligand complexes~Farrow et al, 1994;Stivers et al, 1996;Yu et al, 1996;Zidek et al, 1999!. Taken together with the present results, these studies suggest that regulation of protein entropy may be a rather widespread, albeit subtle, mechanism contributing to a variety of biochemical equilibrium processes.…”

Section: Discussionsupporting

confidence: 77%

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

et al. 2000

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The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15 N NMR-derived order parameters. Longitudinal~R 1 ! and transverse~R 2 ! relaxation rates, transverse cross-relaxation rates~h xy !, and steady state $ 1 H%-15 N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 8C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R 2 0h xy was used to identify nuclei for which conformational exchange makes a significant contribution to R 2 . Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter~S 2 ! was used to calculate the contribution of each NH group to conformational heat capacity~C p ! and a characteristic temperature~T *!, representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ;0.8 kJ0mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ;9 8C lower~78 8C rather than 87 8C!. Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.Keywords: B1 domain; entropy; heat capacity; NMR relaxation; order parameter; protein dynamics; protein stability Most globular proteins are marginally stable because the factors that favor formation of the native state, primarily desolvation of hydrophobic groups and formation of intramolecular hydrogen bonds and salt bridges, are almost equally balanced against those that favor denaturation, primarily the higher conformational entropy of the unfolded protein chain relative to that of the native state~Creigh-ton, 1993; Fersht, 1999!. At high temperatures, the balance between these factors is altered such that many proteins exhibit reversible thermal denaturation with a characteristic midpoint, the melting temperature~T m !. The free energy of unfolding of a proteiñ DG NϪU ! depends upon the changes in enthalpy, entropy, and heat capacity that occur upon unfolding and the temperature~T ! according to the equation~Creighton, 1993; Fersht, 1999!:in which DH 0 and DS 0 are the enthalpy and entropy changes, respectively, at a reference temperature T 0 and DC p,NϪU is the change in heat capacity at constant pressure, which is assumed to be invariant with temperature. Equation 1 indicates that native proteins may be stabilized by an increase in DH 0 or by a decrease in either DS 0 or DC p,NϪU . Thus, one possible strategy for a protein to achieve high thermal ...

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Section: Discussionsupporting

confidence: 77%

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

et al. 2000

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“…Most residues that require the determination of s e to fit the model, with or without solute, are located in the loop. Similar behaviour has been observed in the loop regions or turns of E. coli topoisomerase I [52]. The average s e value for these high frequency motions is also left almost undisturbed by solute addition.…”

Section: Discussionsupporting

confidence: 76%

Protein stabilization by compatible solutes

Lamosa

Turner

Ventura

et al. 2003

European Journal of Biochemistry

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Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin [Lamosa, P., Burke, A., Peist, R., Huber, R., Liu, M.Y., Silva, G., Rodrigues-Pousada, C., LeGall, J., Maycock, C. & Santos, H. (2000) Appl. Environ. Microbiol. 66, 1974Microbiol. 66, -1979. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20°C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJAEmol )1 in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.Keywords: chemical exchange; compatible solutes; protein dynamics; rubredoxin; thermostability.Protein stability, activity and dynamics are interrelated issues with great importance not only in physiological processes but also in protein engineering. The evolution of protein structures towards extreme thermostability was vital for hyperthermophiles, microorganisms thriving near the boiling point of water. In general, the proteins of these organisms are intrinsically resistant to heat denaturation. However, hyperthermophiles also possess intracellular proteins that are not particularly stable, implying the existence of alternative strategies for their stabilization in vivo [1,2]. Hyperthermophiles accumulate high levels of charged organic osmolytes in response to supra-optimal growth temperatures, and this observation led to the hypothesis that these compounds play a role in thermoprotection of macromolecules in vivo [3,4]. This view is supported by in vitro studies showing that these osmolytes protect proteins against heat [1,[5][6][7][8]. Nevertheless, the molecular basis for this well established stabilization phenomenon remains elusive.Several possible mechanisms for protein stabilization by osmolytes have been proposed [9][10][11]. Arakawa and Timasheff [12,13] proposed a preferential hydration model to explain protein stabilization by compatible solutes: solute molecules are excluded from the protein surface, thereby making denaturation entropically less favourable. In conformity, exclusion factors have been measured for a variety of organic solutes and salts [14][15][16], however, the correlation between exclusion factors and the degree of protection ...

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“…decrease in motion (or dynamics) (32). The experimental observation that binding can increase dynamics in some systems (20)(21)(33)(34)(35)(36)(37) emphasizes the importance of entropic contributions. The success of our approach in capturing this effect implies that the entropic contributions are adequately represented in the ensemble view as implemented by the COREX algorithm (6)(7)(8)(9).…”

Section: Resultsmentioning

confidence: 99%

Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble

Pan

Lee

Hilser

2000

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

213

244

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To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase. Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication. First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin. These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity. Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein. Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e.g., binding ligand). The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another. Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation. M ost cellular processes, which are facilitated by proteins, are modulated by effectors. The basic features of such a mechanism of regulation are the presence of multiple binding sites for various ligands and communication between these binding sites, which often are situated many angstroms apart. An understanding of the ground rules of this regulatory mechanism requires a quantitative definition of the functional linkages between these binding sites. In 1964, Wyman introduced the thermodynamic concept of linked functions to establish a quantitative formulation for describing the mutual influence of binding sites on each other (1). Linkage theory is based on thermodynamic principles, is applicable to all biological systems, and exhibits quantitative predictive power. Although Wyman's theory provides the mathematical relationships, it does not address the mechanism through which different binding sites communicate. Thus, besides functional linkage, there are underlying structural-thermodynamic linkages that define the mechanism of site-site communication.Despite a significant body of literature showing that information is transmitted through biological systems via a series of interand intramolecular communication events (refs. 2-5 and references therein), a quantitative predictive theory of structural linkage analogous to the Wyman Linkage Theory is not available. Recently, however, a theoretical approach was established to treat the native state of a protein as an ensemble of conformationa...

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Backbone Dynamics of the C-Terminal Domain of Escherichia coli Topoisomerase I in the Absence and Presence of Single-Stranded DNA

Cited by 78 publications

References 22 publications

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

Protein stabilization by compatible solutes

Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble

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