Uncovering the early stages of domain melting in calmodulin with ultrafast temperature-jump infrared spectroscopy

Lucy Minnes; Gregory M. Greetham; Daniel J. Shaw; Ian P. Clark; Robby Fritzsch; Michael Towrie; Anthony W. Parker; Alistair J. Henry; Richard J. Taylor; Neil T. Hunt

doi:10.1021/acs.jpcb.9b08870

Uncovering the early stages of domain melting in calmodulin with ultrafast temperature-jump infrared spectroscopy

Lucy Minnes, Gregory M. Greetham, Daniel J. Shaw, Ian P. Clark, Robby Fritzsch, Michael Towrie, Anthony W. Parker, Alistair J. Henry, Richard J. Taylor, Neil T. Hunt^*

^*Corresponding author for this work

Physics

Research output: Contribution to journal › Article › peer-review

9 Citations (Scopus)

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Abstract

The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca²⁺, but the structural plasticity of the Ca²⁺-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca²⁺ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.

Original language	English
Pages (from-to)	8733-8739
Number of pages	7
Journal	Journal of Physical Chemistry B
Volume	123
Issue number	41
Early online date	26 Sept 2019
DOIs	https://doi.org/10.1021/acs.jpcb.9b08870
Publication status	Published - 17 Oct 2019

Funding

Funding from STFC is gratefully acknowledged for program access to the Central Laser Facility ULTRA spectrometer. L.M. gratefully acknowledges studentship support from STFC Central Laser Facility and the University of Strathclyde. R.F. gratefully acknowledges studentship support from the University of Strathclyde.

Keywords

calmodulin
CaM
thermal melting

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10.1021/acs.jpcb.9b08870Licence: CC BY 4.0

Minnes-etal-JPCB2019-Uncovering-the-early-stages-of-domain-melting-in-calmodulinFinal published version, 2.54 MBLicence: CC BY 4.0

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title = "Uncovering the early stages of domain melting in calmodulin with ultrafast temperature-jump infrared spectroscopy",

abstract = "The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca2+, but the structural plasticity of the Ca2+-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca2+ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.",

keywords = "calmodulin, CaM, thermal melting",

author = "Lucy Minnes and Greetham, \{Gregory M.\} and Shaw, \{Daniel J.\} and Clark, \{Ian P.\} and Robby Fritzsch and Michael Towrie and Parker, \{Anthony W.\} and Henry, \{Alistair J.\} and Taylor, \{Richard J.\} and Hunt, \{Neil T.\}",

year = "2019",

month = oct,

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doi = "10.1021/acs.jpcb.9b08870",

language = "English",

volume = "123",

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T1 - Uncovering the early stages of domain melting in calmodulin with ultrafast temperature-jump infrared spectroscopy

AU - Minnes, Lucy

AU - Greetham, Gregory M.

AU - Shaw, Daniel J.

AU - Clark, Ian P.

AU - Fritzsch, Robby

AU - Towrie, Michael

AU - Parker, Anthony W.

AU - Henry, Alistair J.

AU - Taylor, Richard J.

AU - Hunt, Neil T.

PY - 2019/10/17

Y1 - 2019/10/17

N2 - The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca2+, but the structural plasticity of the Ca2+-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca2+ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.

AB - The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca2+, but the structural plasticity of the Ca2+-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca2+ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.

KW - calmodulin

KW - CaM

KW - thermal melting

UR - https://www.scopus.com/pages/publications/85073056451

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DO - 10.1021/acs.jpcb.9b08870

M3 - Article

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AN - SCOPUS:85073056451

SN - 1520-6106

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JO - Journal of Physical Chemistry B

JF - Journal of Physical Chemistry B

IS - 41

ER -

Uncovering the early stages of domain melting in calmodulin with ultrafast temperature-jump infrared spectroscopy

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