Linear extrapolation method of analyzing solvent denaturation curves

Pace, C. Nick; Shaw, Kevin L.

doi:10.1002/1097-0134(2000)41:4+<1::aid-prot10>3.0.co;2-2

Cited by 298 publications

(287 citation statements)

References 53 publications

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“…It has been empirically determined that the stability of proteins can be expressed as a linear function of denaturant concentration [13,17]. Thus, the linear extrapolation method is routinely used in protein folding studies.…”

Section: Linear Free-energy Relationshipmentioning

confidence: 99%

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Identification and characterization of protein folding intermediates

Gianni

Ivarsson

Jemth

et al. 2007

Biophysical Chemistry

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In order to understand the mechanism by which a polypeptide chain folds into its functionally active native state it is necessary to characterize in detail all the species accumulated along the pathway.The elusive nature of protein folding intermediates poses their identification and characterization as an extremely difficult task in the protein folding field. In the case of small single domain proteins, the direct measurement of the thermodynamics and structural parameters of protein folding intermediates has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. Here we summarize some of the experimental approaches aimed at the detection and characterization of folding intermediates along with a discussion of some general structural features emerging from these studies.

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Section: Linear Free-energy Relationshipmentioning

confidence: 99%

“…stability in the absence of denaturant by monitoring the fraction of unfolded protein under different denaturant concentrations [17]. The inset panel in Figure 1 shows an example of how such linear relationship can be employed using a classical co-operative unfolding profile.…”

Section: Linear Free-energy Relationshipmentioning

confidence: 99%

Identification and characterization of protein folding intermediates

Gianni

Ivarsson

Jemth

et al. 2007

Biophysical Chemistry

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“…The data were analyzed via the Linear Extrapolation Method of Greene and Pace via nonlinear least squares (36,37) as proposed by Santoro and Bolen (38). The parameters used to fit the data are slope and intercept for the pretransition region, slope, and intercept for the posttransition region, and two or three parameters to describe the free energy change of unfolding, i.e.,…”

Section: Methodsmentioning

confidence: 99%

Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Peng

Veneziano

Gillispie

et al. 2010

Journal of Biological Chemistry

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“…The product of m eq and C 1/2 yields the equilibrium standard free energy of denaturation (∆G N-D ) at 0 M urea, calculated using the linear extrapolation method (43).…”

Section: Molecularmentioning

confidence: 99%

Kinetic Role of Helix Caps in Protein Folding Is Context-Dependent

2004

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Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of R-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33;Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641. We have made site-specific substitutions in the N-and C-cap motifs of the 5-helix protein monomeric λ repressor (λ 6-85 ) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly contextdependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.The native and denatured conformational ensembles involved in the process of protein folding are important in proper cellular function. If the native state is the functional species, then that function is lost during any time spent in the unfolded state or in the folding process. The denatured or unfolded ensemble of structures may be more susceptible to protein turnover through cellular degradation pathways. Additionally, proper cellular localization and protein trafficking is often coupled to the folding process. A detailed understanding of the medically important process of protein misfolding and aggregation requires better molecular characterization of kinetic folding mechanisms.Each residue in a protein does not have an equal role in the folding process. Several studies have shown that often only a small subset of residues attain nativelike structure in the transition state (3-6). In this paper, we consider the role of helix termini in protein folding by determining the kinetic consequences of site-specific substitutions in helix N-and C-cap structural motifs.Helix N-and C-cap residues are found at the beginning and end of helices, ending the regular i to i + 4 hydrogen bond pattern ...

show abstract

Linear extrapolation method of analyzing solvent denaturation curves

Cited by 298 publications

References 53 publications

Identification and characterization of protein folding intermediates

Identification and characterization of protein folding intermediates

Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Kinetic Role of Helix Caps in Protein Folding Is Context-Dependent

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