Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations

J. Evenäs, S. Forsén, A. Malmendal, M. Akke

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between 'open' and 'closed' conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of15N spin relaxation experiments at three this mutant were studied using different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7 x 103s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2 x 104to 3 x 104s-1at 291 K for a subset of residues. The temperature dependence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.
OriginalsprogEngelsk
TidsskriftJournal of Molecular Biology
Vol/bind289
Udgave nummer3
ISSN0022-2836
DOI
StatusUdgivet - 1999

Emneord

  • Conformational exchange
  • Cross-correlation relaxation rates
  • Energy barriers
  • NMR spin relaxation
  • Order parameters

Citer dette

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title = "Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations",
abstract = "Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between 'open' and 'closed' conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of15N spin relaxation experiments at three this mutant were studied using different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7 x 103s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2 x 104to 3 x 104s-1at 291 K for a subset of residues. The temperature dependence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.",
keywords = "Conformational exchange, Cross-correlation relaxation rates, Energy barriers, NMR spin relaxation, Order parameters",
author = "J. Even{\"a}s and S. Fors{\'e}n and A. Malmendal and M. Akke",
year = "1999",
doi = "10.1006/jmbi.1999.2770",
language = "English",
volume = "289",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
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Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations. / Evenäs, J.; Forsén, S.; Malmendal, A.; Akke, M.

I: Journal of Molecular Biology, Bind 289, Nr. 3, 1999.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations

AU - Evenäs, J.

AU - Forsén, S.

AU - Malmendal, A.

AU - Akke, M.

PY - 1999

Y1 - 1999

N2 - Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between 'open' and 'closed' conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of15N spin relaxation experiments at three this mutant were studied using different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7 x 103s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2 x 104to 3 x 104s-1at 291 K for a subset of residues. The temperature dependence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.

AB - Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between 'open' and 'closed' conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of15N spin relaxation experiments at three this mutant were studied using different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7 x 103s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2 x 104to 3 x 104s-1at 291 K for a subset of residues. The temperature dependence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.

KW - Conformational exchange

KW - Cross-correlation relaxation rates

KW - Energy barriers

KW - NMR spin relaxation

KW - Order parameters

U2 - 10.1006/jmbi.1999.2770

DO - 10.1006/jmbi.1999.2770

M3 - Journal article

VL - 289

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -