Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate

Marc Jamin, Syun-Ru Yeh, Denis L. Rousseau, Robert L. Baldwin

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia ⇆ Ib. In urea-induced unfoIding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib→Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U ⇆ Ia ⇆ N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2.7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Original languageEnglish (US)
Pages (from-to)731-740
Number of pages10
JournalJournal of Molecular Biology
Volume292
Issue number3
DOIs
StatePublished - Sep 24 1999

Fingerprint

Sperm Whale
Protein Unfolding
Fluorescence Spectrometry
Tryptophan
Urea
apomyoglobin
Acids

Keywords

  • Apomyoglobin
  • Cooperativity
  • Folding intermediate
  • Molten globule
  • Tryptophan fluorescence

ASJC Scopus subject areas

  • Virology

Cite this

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate. / Jamin, Marc; Yeh, Syun-Ru; Rousseau, Denis L.; Baldwin, Robert L.

In: Journal of Molecular Biology, Vol. 292, No. 3, 24.09.1999, p. 731-740.

Research output: Contribution to journalArticle

@article{8fc09dceca2a4b3d9b0706098a50dae6,
title = "Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate",
abstract = "Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia ⇆ Ib. In urea-induced unfoIding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib→Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U ⇆ Ia ⇆ N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2.7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.",
keywords = "Apomyoglobin, Cooperativity, Folding intermediate, Molten globule, Tryptophan fluorescence",
author = "Marc Jamin and Syun-Ru Yeh and Rousseau, {Denis L.} and Baldwin, {Robert L.}",
year = "1999",
month = "9",
day = "24",
doi = "10.1006/jmbi.1999.3074",
language = "English (US)",
volume = "292",
pages = "731--740",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate

AU - Jamin, Marc

AU - Yeh, Syun-Ru

AU - Rousseau, Denis L.

AU - Baldwin, Robert L.

PY - 1999/9/24

Y1 - 1999/9/24

N2 - Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia ⇆ Ib. In urea-induced unfoIding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib→Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U ⇆ Ia ⇆ N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2.7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

AB - Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia ⇆ Ib. In urea-induced unfoIding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib→Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U ⇆ Ia ⇆ N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2.7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

KW - Apomyoglobin

KW - Cooperativity

KW - Folding intermediate

KW - Molten globule

KW - Tryptophan fluorescence

UR - http://www.scopus.com/inward/record.url?scp=0033600716&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033600716&partnerID=8YFLogxK

U2 - 10.1006/jmbi.1999.3074

DO - 10.1006/jmbi.1999.3074

M3 - Article

C2 - 10497035

AN - SCOPUS:0033600716

VL - 292

SP - 731

EP - 740

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -