Conformational Stability of the <i>Escherichia coli</i> HPr Protein:  Test of the Linear Extrapolation Method and a Thermodynamic Characterization of Cold Denaturation

Em, Nicholson; Jm, Scholtz

doi:10.1021/bi960863y

Cited by 126 publications

(182 citation statements)

References 44 publications

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“…suggest an even higher T S .! There is no clear trend in how DC p varies with GdnHCl concentration~Pfeil & Privalov, 1976;Makhatadze & Privalov, 1992;Barone et al, 1994;Grantcharova & Baker, 1997;Kuhlman & Raleigh, 1998!, or urea concentration~Pace & Tanford, 1968Griko & Privalov, 1992;Makhatadze & Privalov, 1992;Barone et al, 1994;Scholtz, 1995;Nicholson & Scholtz, 1996;Chiti et al, 1998 Figure 1A.…”

Section: Discussionmentioning

confidence: 99%

Heat capacity change for ribonuclease A folding

et al. 1999

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The change in heat capacity DC p for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, DC p ϭ 1.15 6 0.08 kcal mol Ϫ1 K Ϫ1 . Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method. ⌬C p ϭ d~⌬H !0d~T !1 ! to estimate DC p , and the results are shown in Figure 1. The resulting DC p values are given in Table 2. DC p values based on plots of DH vH and DH fit from the DSC data, a global fit of all of the DSC data, and on the difference between the posttransition C p~D ! and pretransition C p~N ! baselines of the DSC experiments are also given. The DC p value based on plots of DH cal vs. Keywords

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Section: Discussionmentioning

confidence: 99%

Heat capacity change for ribonuclease A folding

et al. 1999

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“…Thermal denaturation curves were fitted to standard equations describing a two-state transition, assuming the excess heat capacity for unfolding (⌬C P D-N ) to be temperature independent (15). Thermal melts were highly reproducible, with the thermal midpoint (T m ) and the enthalpy for unfolding (⌬H D-N ) typically varying by less than 1.2 K and 9 kJ͞mol, respectively, in repeat measurements.…”

Section: Methodsmentioning

confidence: 99%

Ultrafast folding of WW domains without structured aromatic clusters in the denatured state

Ferguson

Johnson

Macias

et al. 2001

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

121

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Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a threestranded antiparallel ␤-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38 -44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 s at 25°C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.T he combination of high-resolution experimental data with detailed computer simulation is required to solve the pathway of protein folding at atomic resolution. Unfortunately, there is an incompatibility between the time regime for folding and unfolding of most proteins [the millisecond to second range (1)] and that of current molecular dynamics (MD) simulations, which are typically conducted at high temperatures (Ͼ200°C) and last for less than 1 s (2). Thus, the folding and unfolding pathways of the majority of real proteins are currently not testable by MD simulations under physiological conditions, although the ␣-helical engrailed homeodomain folds and unfolds so rapidly that simulations could be performed close to experimentally accessible conditions (3).To continue this benchmarking of simulation with experiment, we are studying the all-␤-sheet WW domains, a large family of small single-domain proteins (4) (typically 38-44 amino acids in length) that contain a distorted three-stranded antiparallel ␤-sheet (5). In contrast to all ␣-helical proteins, where the secondary structure is defined more by local interactions and the rules governing folding and stability are fairly well understood (6), analogous processes in ␤-sheet proteins are less clearly defined. As a number of WW domains have well defined monomeric native structures (7-9) that lack cofactors, prosthetic groups, disulfide linkages, and cis prolines that can complicate kinetic analyses, these domains are suitable model systems for understanding the different factors that contribute to ␤-sheet folding and stability.Previous studies focused on a 57-residue human Yes kinase associated protein (YAP 65), where folding and unfolding are reversible and very rapid (10). Evidence was presented for a residual aromatic cluster in the chemically, but not the thermally, denatured state (11). To circumvent potential problems that may be associated wi...

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“…Repeat scans were highly reversible with 95% of the signal recovered after thermal denaturation. Thermal melts were fitted to a two-state transition, with the change in heat capacity upon unfolding (⌬Cp DϪN ) assumed to be temperature-independent (17) MD Simulations. The simulations were performed with the program ENCAD (23) and a previously described force field (24,25).…”

Section: Methodsmentioning

confidence: 99%

Using flexible loop mimetics to extend Φ-value analysis to secondary structure interactions

Ferguson

Pires

Toepert

et al. 2001

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

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Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, ␤-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal͞mol (1 cal ‫؍‬ 4.18 J). Preliminary ⌽-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, ⌽-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions. U nderstanding how proteins spontaneously fold to a stable three-dimensional structure is exceptionally difficult. This difficulty applies especially to ␤-sheet proteins, whose secondary structure is stabilized more by long-range sequence-distant interactions than is that in ␣-helical proteins. Small ␤-sheet proteins are useful paradigms for understanding the search problem. There is increasing evidence of the importance of ␤-hairpins and loops in driving ␤-sheet folding (1-6). The stability of these loops may also influence the overall stability of ␤-sheet proteins in two ways. One way is by contributing to the overall thermodynamic stability of the system (7-9). The other way is in the effect on the folding pathway (2, 3), such that folding may or may not be facilitated by loop or hairpin formation, with concomitant effects on the folding rate and, thus, on stability.The WW domains are a family of small, triple-stranded, antiparallel ␤-sheet proteins (typically, of 34-44 amino acids) with a small hydrophobic core involving residues from both the N and C termini (10). The loop structures and overall bend of the ␤-sheet are well conserved in the different members of this family (11-13). A number of these domains are well suited for biophysical studies of ␤-sheet folding and loop formation as they reversibly denature, are amenable to mutagenesis, and are easily synthesized by using both recombinant and organic chemistry techniques (12,14,15). Thus, these domains can be synthesized with both natural and nonnatural amino acids in their sequences, allowing for sophisticated experiments that cannot easily be performed with recombinant proteins. The effects of substitutions on the stabilities of WW domains have been studied in the context of a designed WW-domain...

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Conformational Stability of the Escherichia coli HPr Protein: Test of the Linear Extrapolation Method and a Thermodynamic Characterization of Cold Denaturation

Cited by 126 publications

References 44 publications

Heat capacity change for ribonuclease A folding

Heat capacity change for ribonuclease A folding

Ultrafast folding of WW domains without structured aromatic clusters in the denatured state

Using flexible loop mimetics to extend Φ-value analysis to secondary structure interactions

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