Φ‐Value Analysis of Protein Folding Transition States

Ferguson, Neil; Fersht, Alan R.

doi:10.1002/9783527634026.ch4

Cited by 5 publications

(5 citation statements)

References 87 publications

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“…Conservative point mutations were used that should not significantly alter the structure of D (26). Thus, significant free energy changes most likely report on changes in the relative stability of N and I (26,49).…”

Section: Resultsmentioning

confidence: 99%

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

Alexander

Jürgens

Shepherd

et al. 2013

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

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Significance Hepatitis B virus (HBV) is a major pathogen, yet no fully effective therapies exist. HBc is the multifunctional, capsid-forming protein essential for HBV replication. HBc structural plasticity is reportedly functionally important. We analyzed the folding mechanism of HBc using a multidisciplinary approach, including microscale thermophoresis and ion mobility spectrometry–mass spectrometry. HBc folds in a 3-state transition with a dimeric, helical intermediate. We found evidence of a strained native ensemble wherein the energy landscapes for folding, allostery, and capsid formation are linked. Mutations thermodynamically trapped HBc in conformations unable to form capsids, suggesting chemical chaperones could elicit similar, potentially antiviral, effects.

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Section: Resultsmentioning

confidence: 99%

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

Alexander

Jürgens

Shepherd

et al. 2013

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

Self Cite

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“…This approach has been successfully used in protein folding studies, and the basic idea is to compare changes in respectively binding-and activation energy. Principles and practices of the approach have been lucidly discussed elsewhere (58,59), and will not be reiterated here, except for some key aspects of the current application, which are sketched out in Fig. 5.…”

Section: Trp-variants and φ-Factor Analysismentioning

confidence: 99%

Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues

Røjel

Kari

Sørensen

et al. 2020

Journal of Biological Chemistry

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Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 105m−1 s−1 and 5 × 10−3 s−1, respectively, reflecting the slow dynamics of a solid, polymeric ligand. Especially the off-rate constant was many orders of magnitude lower than typical values for small, soluble ligands. Binding rate and strength both were typically lower for the Trp variants, but effects of the substitutions were moderate and sometimes negligible. Hence, we propose that lowering the activation barrier for complexation is not a major driving force for the high conservation of the Trp residues. Using so-called Φ-factor analysis, we analyzed the kinetic and thermodynamic results for the variants. The results of this analysis suggested a transition state for complexation and decomplexation in which the reducing end of the ligand is close to the tunnel entrance (near Trp-40), whereas the rest of the binding tunnel is empty. We propose that this structure defines the highest free-energy barrier of the overall catalytic cycle and hence governs the turnover rate of this industrially important enzyme.

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“…We determined whether variations in the ionic strength or pH modulated the stability of SAP L31W (Fig. 4 A and B, filled circles) in order to identify the optimal conditions for in vitro studies of protein folding reactions ( Ferguson and Fersht, 2008 ). Ideally, such studies should be performed under conditions where the protein stability is independent of small changes in pH, so that changes in pH with temperature (arising from non-zero ionisation enthalpies for buffers) or batch-to-batch variation in reagents have negligible effects.…”

Section: Resultsmentioning

confidence: 99%

Engineering a two-helix bundle protein for folding studies

Dodson¹,

Ferguson²,

Rutherford³

et al. 2010

Protein Engineering Design and Selection

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The SAP domain from the Saccharomyces cerevisiae THO1 protein contains a hydrophobic core and just two α-helices. It could provide a system for studying protein folding that bridges the gap between studies on isolated helices and those on larger protein domains. We have engineered the SAP domain for protein folding studies by inserting a tryptophan residue into the hydrophobic core (L31W) and solved its structure. The helical regions had a backbone root mean-squared deviation of 0.9 Å from those of wild type. The mutation L31W destabilised wild type by 0.8 ± 0.1 kcal mol−1. The mutant folded in a reversible, apparent two-state manner with a microscopic folding rate constant of around 3700 s−1 and is suitable for extended studies of folding.

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Φ‐Value Analysis of Protein Folding Transition States

Cited by 5 publications

References 87 publications

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues

Engineering a two-helix bundle protein for folding studies

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