Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis

Equilibria with high kinetic barriers

Zhihong Lai, Jennifer McCulloch, Hilal A. Lashuel, Jeffery W. Kelly

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

Fluorescence and circular dichroism spectroscopy as well as analytical ultracentrifugation and glutaraldehyde cross-linking were utilized to evaluate the tertiary and quaternary structural changes occurring on the denaturation and reconstitution pathways of transthyretin (TTR) as a function of guanidine hydrochloride (GdnHCl) concentration. These results demonstrate that the GdnHCl-mediated denaturation and reconstitution of TTR is reversible. However, the lowest GdnHCl concentration that dissociates and unfolds transthyretin does not allow the unfolded monomer to refold to tetramer at a rate that is measurable. As a result, there is a striking hysteresis observed upon comparison of the GdnHCl-mediated denaturation and reconstitution transitions. The TTR tetramer does not dissociate into unfolded monomer until the denaturant concentration exceeds 4 M GdnHCl, whereas unfolded monomeric TTR (denatured in 7 M GdnHCl) does not refold and assemble into a native tetrameric structure until the GdnHCl concentration is reduced to less than 2 M. These results imply that a significant kinetic barrier intervenes between the folded tetramer and unfolded monomer in both the denaturation and reconstitution directions at pH 7. A kinetics study of the denaturation of TTR as a function of GdnHCl concentration yields a first- order rate constant for unfolding of (9.0 ± 7.5) x 10-11 s-1, estimated by extrapolation of the rate constants for the tetramer to unfolded monomer transition as a function of GdnHCl to 0 M GdnHCl. This rate is very slow; as a result, wild-type TTR is predicted to be kinetically stable as a tetrameric quaternary structure once formed. These results imply that the rate of TTR dissociation and partial unfolding to the monomeric amyloidogenic intermediate under denaturing conditions may play a role in transthyretin- based amyloid diseases.

Original languageEnglish
Pages (from-to)10230-10239
Number of pages10
JournalBiochemistry
Volume36
Issue number33
DOIs
Publication statusPublished - 19 Aug 1997
Externally publishedYes

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Denaturation
Prealbumin
Guanidine
Hysteresis
Kinetics
Monomers
Rate constants
Circular dichroism spectroscopy
Ultracentrifugation
Glutaral
Circular Dichroism
Amyloid
Extrapolation
Spectrum Analysis
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis : Equilibria with high kinetic barriers. / Lai, Zhihong; McCulloch, Jennifer; Lashuel, Hilal A.; Kelly, Jeffery W.

In: Biochemistry, Vol. 36, No. 33, 19.08.1997, p. 10230-10239.

Research output: Contribution to journalArticle

Lai, Zhihong ; McCulloch, Jennifer ; Lashuel, Hilal A. ; Kelly, Jeffery W. / Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis : Equilibria with high kinetic barriers. In: Biochemistry. 1997 ; Vol. 36, No. 33. pp. 10230-10239.
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abstract = "Fluorescence and circular dichroism spectroscopy as well as analytical ultracentrifugation and glutaraldehyde cross-linking were utilized to evaluate the tertiary and quaternary structural changes occurring on the denaturation and reconstitution pathways of transthyretin (TTR) as a function of guanidine hydrochloride (GdnHCl) concentration. These results demonstrate that the GdnHCl-mediated denaturation and reconstitution of TTR is reversible. However, the lowest GdnHCl concentration that dissociates and unfolds transthyretin does not allow the unfolded monomer to refold to tetramer at a rate that is measurable. As a result, there is a striking hysteresis observed upon comparison of the GdnHCl-mediated denaturation and reconstitution transitions. The TTR tetramer does not dissociate into unfolded monomer until the denaturant concentration exceeds 4 M GdnHCl, whereas unfolded monomeric TTR (denatured in 7 M GdnHCl) does not refold and assemble into a native tetrameric structure until the GdnHCl concentration is reduced to less than 2 M. These results imply that a significant kinetic barrier intervenes between the folded tetramer and unfolded monomer in both the denaturation and reconstitution directions at pH 7. A kinetics study of the denaturation of TTR as a function of GdnHCl concentration yields a first- order rate constant for unfolding of (9.0 ± 7.5) x 10-11 s-1, estimated by extrapolation of the rate constants for the tetramer to unfolded monomer transition as a function of GdnHCl to 0 M GdnHCl. This rate is very slow; as a result, wild-type TTR is predicted to be kinetically stable as a tetrameric quaternary structure once formed. These results imply that the rate of TTR dissociation and partial unfolding to the monomeric amyloidogenic intermediate under denaturing conditions may play a role in transthyretin- based amyloid diseases.",
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