Thermodynamics of Denaturation of Barstar: Evidence for Cold Denaturation and Evaluation of the Interaction with Guanidine Hydrochloride

Agashe, Vishwas R.; Udgaonkar, Jayant B.

doi:10.1021/bi00010a019

Cited by 215 publications

(295 citation statements)

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“…Several studies have examined the effects of cosolvents on ⌬H°, ⌬S°, and ⌬C p for protein unfolding (Pfeil and Privalov 1976;Makhatadaze and Privalov 1992;Agashe and Udgaonkar 1995;DeKoster and Robertson 1997;Zweifel and Barrick 2002). Makhatadze and Privalov (1992) examined the thermodynamics of interaction of urea and guanidine with native and denatured proteins and found significant effects of these cosolvents on both enthalpy and entropy of interaction.…”

Section: The Relationship Between the Influence Of A Cosolvent On T Mmentioning

confidence: 99%

Measuring the stability of partly folded proteins using TMAO

2003

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Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that ⌬G°for unfolding depends linearly on TMAO concentration, and that the sensitivity of ⌬G°to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of ⌬G°on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.Keywords: Protein stability; protein folding; Notch ankyrin domain; Barnase; osmolytes Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is found in several marine organisms containing elevated intracellular urea concentrations (Robertson 1966(Robertson , 1975Griffith et al. 1974). Numerous studies have investigated the effect of TMAO on proteins and described its stabilizing effects (Yancey and Somero 1979;Lin and Timasheff 1994;Jaravine et al. 2000). Yancey et al. (1982) showed, by gel filtration chromatography, that TMAO promotes folding of proteins into more compact forms. They further showed, by recovery of enzymatic activity, that TMAO promotes folding to specific, biologically relevant native states (Yancey et al. 1982). Wang and Bolen (1997) provided an explanation for the ability of TMAO to promote specific refolding to the native structure, in which unfavorable thermodynamic interactions between TMAO and the peptide backbone destabilize the denatured state, shifting equilibrium toward the native state. Preferential interaction data from Lin and Timasheff (1994) are consistent with this interpretation, showing the region of solvent near the denatured state of the protein to be rarified in TMAO.The functional dependence of protein stability on TMAO has been analyzed in several systems and has led to different Reprint requ...

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Section: The Relationship Between the Influence Of A Cosolvent On T Mmentioning

confidence: 99%

Measuring the stability of partly folded proteins using TMAO

2003

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“…Although the energetics of barnase have been studied in detail by several groups (Hartley, 1%8, 1975; Makarov et al, 1993;Griko et al, 1994;Sanz et al, 1994), the energetics of native barstar are less well understood. The free energy of barstar unfolding by urea was estimated by Schreiber and Fersht (1993b), and the van't Hoff enthalpy of barstar unfolding was recently estimated by Agashe and Udgaonkar (1995). However, the energetics of barstar unfolding have yet to be measured in a direct, modelindependent manner.…”

Section: Abstract: Barstar; Protein Stability; Scanning Microcalorimetrymentioning

confidence: 99%

Structural energetics of barstar studied by differential scanning microcalorimetry

1995

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The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at =75 "C was found to be 5.6 f 0.3 kJ K" mol". In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 f 0.03 cal K-' gg', which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase. Our data, which indicate that barstar has a loosely packed interior, suggest that high conformational flexibility of barstar's native structure may play an important role in allowing it to optimize its contacts with barnase upon binding without disrupting favorable, tightly packed internal interactions. Keywords: barstar; protein stability; scanning microcalorimetryThe interaction between barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its natural inhibitor, has attracted a great deal of attention as a good model for the study of protein-protein recognition (Hartley, 1993;Schreiber & Fersht, 1993a). The crystal structure of Cys 40, 82 Ala barstar complexed with barnase was recently solved with a resolution that is sufficient for detailed analysis of the interactions stabilizing the complex (Guillet et al., 1993;Buckle et al., 1994), and the structures of both free barnase (Mauguen et al., 1982;Bycroft et al., 1991) and free barstar (Lubienski et al., 1994) are known. To understand the mechanisms by which these proteins associate with one another, we require knowledge of the thermodynamics of complex formation and the energetics of native structure formation in both free barnase and free barstar. Although the energetics of barnase have been studied in detail by several groups (Hartley, 1%8, 1975; Makarov et al., 1993;Griko et al., 1994;Sanz et al., 1994), the energetics of native barstar are less well understood. The free energy of barstar unfolding by urea was estimated by Schreiber and Fersht (1993b), and the van't Ho...

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“…However, rapid CO rebinding, binding of non-native ligands, and possibly aggregation prevented the complete transition to the native state (11). A more generally applicable method for studying fast events would be to raise rapidly the temperature of a cold-denatured protein, since cold denaturation is a common phenomenon of globular proteins under suitable experimental conditions (12)(13)(14)(15). Temperature jump (T-jump) by electrical discharge (16)(17)(18)(19)(20) and laser-induced heating would thus enable microsecond and nanosecond time resolutions, respectively.…”

mentioning

confidence: 99%

Submillisecond events in protein folding.

Nölting

Golbik

Fersht

1995

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

138

122

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The pathway of protein folding is now being analyzed at the resolution of individual residues by kinetic measurements on suitably engineered mutants. The kinetic methods generally employed for studying folding are typically limited to the time range of 21 ms because the folding of denatured proteins is usually initiated by mixing them with buffers that favor folding, and the dead time of rapid mixing experiments is about a millisecond. We now show that the study of protein folding may be extended to the microsecond time region by using temperature-jump measurements on the cold-unfolded state of a suitable protein. We are able to detect early events in the folding of mutants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and 4D-value analysis indicates that it is a transition between two relatively solventexposed states with little consolidation of structure.The pathway by which a particular protein folds will be resolved experimentally when the structures of all stable, metastable, and transition states adopted by the protein during the process have been characterized structurally and energetically. The only way to analyze the structures of transition states is by kinetics, and the details of their structure and energetics can be gleaned from the kinetics of folding of proteins whose structures have been carefully altered by protein engineering (1-6). This protein engineering procedure has been used to characterize transition states and intermediates on a time scale of .1 ms (1-6), the time resolution of the stopped-flow and other rapid mixing techniques employed so far in these and most other studies (e.g., refs. 7-9). This is too slow to allow detection of the early events that initiate folding. To study faster events, kinetic techniques must be employed that eliminate the time delays arising from rapid mixing techniques. Relaxation methods, by which a preexisting equilibrium between denatured and folded states is rapidly perturbed by a change in physical or chemical conditions, may be used to extend the time range. It is not easy, however, to find methods that can readily cause a denatured state of a protein to renature. Millisecond time resolution has been achieved by using a repetitive pressure-perturbation method (10). Laser flash photolysis has been applied to dissociate CO from CO-bound cytochrome c, with concomitant denaturation (11). However, rapid CO rebinding, binding of non-native ligands, and possibly aggregation prevented the complete transition to the native state (11). A more generally applicable method for studying fast events would be to raise rapidly the temperature of a cold-denatured protein, since cold denaturation is a common phenomenon of globular proteins under suitable experimental conditions (12-15). Temperature jump (T-jump) by electrical discharge (16-20) and laser-induced heating would thus enable microsecond and nanosecond time resolutions, respectively.The structure of barstar (21), the inhibitor of th...

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Thermodynamics of Denaturation of Barstar: Evidence for Cold Denaturation and Evaluation of the Interaction with Guanidine Hydrochloride

Cited by 215 publications

References 40 publications

Measuring the stability of partly folded proteins using TMAO

Measuring the stability of partly folded proteins using TMAO

Structural energetics of barstar studied by differential scanning microcalorimetry

Submillisecond events in protein folding.

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