Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

Zhu, Li; Fan, Yi; Zhou, Jun‐Mei

doi:10.1016/s0167-4838(00)00244-2

Cited by 29 publications

(21 citation statements)

References 38 publications

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“…One is that the reactions display hysteresis between assembly and disassembly because of the high energetic barrier to disassembly 34 . Hysteresis is often seen in association reactions of large, multidomain proteins such as bacterial luciferase 33 , transthyretin 32 , creatine kinase 44 , and collagen 45 . All of these examples are marked by high kinetic energy barriers to complex dissociation, which prevent coincidence of unfolding/folding transitions in experimentally convenient time scales; e.g., ~ a million years for the dissociation of bacterial luciferase.…”

Section: Hysteresis In Assembly Reactionsmentioning

confidence: 99%

Phage P22 Procapsids Equilibrate with Free Coat Protein Subunits

Parent

Suhanovsky

Teschke

2007

Journal of Molecular Biology

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Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been treated as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsids assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure coat protein monomers are mixed with scaffolding protein. Here we present data that show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding protein stripped out) were diluted so that the concentration was below the dissociation constant (~5 μM) for coat protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented here illustrate that coat protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and follows the law of mass action.

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Section: Hysteresis In Assembly Reactionsmentioning

confidence: 99%

Phage P22 Procapsids Equilibrate with Free Coat Protein Subunits

Parent

Suhanovsky

Teschke

2007

Journal of Molecular Biology

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“…Chemical denaturants, such as guanidine hydrochloride and urea, have been widely used in the investigations of the unfolding of CK (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Because there is a direct interaction between a denaturant and a protein, some thermodynamic parameters may reflect protein-denaturant interaction rather than intrinsic parameters of the protein (15).…”

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confidence: 99%

Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid

Liang

Sanglier

et al. 2003

Journal of Biological Chemistry

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Creatine kinase (CK, 1 EC 2.7.3.2) is a key enzyme for energy homeostasis in cells and plays a significant role in the transport of high energy phosphates via phosphocreatine to sites of ATP utilization in vivo (1-4). This enzyme catalyzes the reversible phosphoryl transfer between ATP and creatine (Cr) in the presence of Mg 2ϩ , and the release of an equimolar quantity of hydrogen ion (see Reaction I).Cr ϩ MgATP 2Ϫ º PCr 2Ϫ ϩ MgADP Ϫ ϩ H ϩ REACTION I

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“…Previous investigation suggests that addition of glycerol can slow down the rate of subsequent folding after the burst phase and prevent the formation of the off-pathway intermediate [16]. Under these conditions (at lower protein concentrations), the refolded state populated at equilibrium is spectroscopically indistinguishable from the native state and exhibits greater than 50% native activity, suggesting a native-like structure.…”

Section: Time-dependent Refolding From 8 M Urea-denatured Ck Monitorementioning

confidence: 88%