Thermodynamic Analysis of the Low- to Physiological-Temperature Nondenaturational Conformational Change of Bovine Carbonic Anhydrase

Hollowell, Heather; Younvanich, Saronya S; McNevin, Stacey L.; Britt, B. Mark

doi:10.5483/bmbrep.2007.40.2.205

Cited by 8 publications

(7 citation statements)

References 16 publications

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“…Reactions at all four cysteines were enhanced by raising the temperature to 37°C, but the effect varied from about two-fold for a cysteine at 338 to greater than seven-fold for a cysteine at 337. As expected, the apparent activation energies varied widely, ranging from being that expected for disulfide exchange (15,16) for T338C CFTR to values in the range of those generally associated with protein conformational change (17,18,19,20). …”

Section: Resultssupporting

confidence: 58%

Cystic Fibrosis Transmembrane Conductance Regulator: Temperature-Dependent Cysteine Reactivity Suggests Different Stable Conformers of the Conduction Pathway

Liu

Dawson

2011

Biochemistry

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Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6), prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22°C to 37°C for cysteines placed on either side of an apparent size-selective, accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336 and 337, is highly temperature dependent, such that at 37°C cysteines at these positions were highly reactive toward MTSES−, whereas at 22°C the reaction rates ranged from two to six-fold slower to undetectable. An activation energy of 157 kJ/mole for the reaction at 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those accessible at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

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Section: Resultssupporting

confidence: 58%

Cystic Fibrosis Transmembrane Conductance Regulator: Temperature-Dependent Cysteine Reactivity Suggests Different Stable Conformers of the Conduction Pathway

Liu

Dawson

2011

Biochemistry

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“…The latest research on the structure and function of CA has focused on analyzing the proton shuttle, ,− ,− the binding of zinc to CA, and the p K a of the zinc-bound water . In addition, several groups have further examined the denaturation, aggregation, and folding of CA. − …”

Section: 6 Codamentioning

confidence: 99%

“…305 In addition, several groups have further examined the denaturation, aggregation, and folding of CA. [875][876][877][878] Our hope is that this review will be useful to investigators who are looking for a way to navigate this still very dynamic field. Mechanism of catalysis of the hydration of CO 2 by HCA II.…”

mentioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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“…However, barriers of this magnitude are characteristic of processes that typically do not occur on biological time scales. 7,8 Although the aforementioned experimental studies imply that partially unfolded states of collagen do not occur spontanesously, 4,6 other studies suggest that collagen is conformationally labile in the vicinity of the collagenase cleavage site. 3,9-12 Accordingly, one alternate theory is that collagen exists as an equilibrium distribution of conformational states, where some states are partially unfolded in the vicinity of the collagenase cleavage site.…”

Section: Introductionmentioning

confidence: 99%

“…This significant barrier exists when the enzyme is present. However, barriers of this magnitude are characteristic of processes that typically do not occur on biological time scales 7, 8…”

Section: Introductionmentioning

confidence: 99%

Do collagenases unwind triple‐helical collagen before peptide bond hydrolysis? Reinterpreting experimental observations with mathematical models

Nerenberg¹,

Salsas-Escat²,

Stultz

2007

Proteins

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It has been postulated that triple-helical collagen is actively unwound by collagenases before peptide bond hydrolysis--a supposition that explains the small catalytic rate constant associated with collagenolysis. We propose an alternate model of collagen degradation that does not require active unwinding by collagenases, but instead suggests that the regions of collagen near the collagenase cleavage site can adopt either a native triple-helical or a partially unfolded conformation. In this model, collagenases preferentially bind to and stabilize partially unfolded conformers before cleaving the scissile bond. Existing experimental observations (which were previously taken to support active unwinding models) are reinterpreted using corroborative evidence from numerical simulations and found to be consistent with this framework. These data support the notion that collagen, like all other biological heteropolymers, undergoes thermal fluctuations that cause it to sample distinct structures in the neighborhood of the native state, and collagenolysis occurs when collagenases recognize the appropriate unwound conformers.

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Thermodynamic Analysis of the Low- to Physiological-Temperature Nondenaturational Conformational Change of Bovine Carbonic Anhydrase

Cited by 8 publications

References 16 publications

Cystic Fibrosis Transmembrane Conductance Regulator: Temperature-Dependent Cysteine Reactivity Suggests Different Stable Conformers of the Conduction Pathway

Cystic Fibrosis Transmembrane Conductance Regulator: Temperature-Dependent Cysteine Reactivity Suggests Different Stable Conformers of the Conduction Pathway

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Do collagenases unwind triple‐helical collagen before peptide bond hydrolysis? Reinterpreting experimental observations with mathematical models

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