Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy

Lucy Minnes, Daniel J. Shaw, Benjamin P. Cossins, Paul M. Donaldson, Gregory M. Greetham, Michael Towrie, Anthony W. Parker, Matthew J. Baker, Alistair J. Henry, Richard J. Taylor, Neil T. Hunt

Research output: Contribution to journalArticle

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Abstract

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally-stable Ca2+-bound (holo-) CaM. The functionally-relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.
LanguageEnglish
Pages10898-10906
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number20
Early online date18 Sep 2017
DOIs
StatePublished - 17 Oct 2017

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Calmodulin
Infrared spectroscopy
Molecular dynamics
Melting
Circular dichroism spectroscopy
Structural dynamics
Computer simulation
Dichroism
Conformations
Proteins
Calcium
Heating
Amino Acids
Temperature

Keywords

  • spectroscopy
  • data analysis
  • structure
  • Calmodulin

Cite this

Minnes, L., Shaw, D. J., Cossins, B. P., Donaldson, P. M., Greetham, G. M., Towrie, M., ... Hunt, N. T. (2017). Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy. Analytical Chemistry, 89(20), 10898-10906. DOI: 10.1021/acs.analchem.7b02610
Minnes, Lucy ; Shaw, Daniel J. ; Cossins, Benjamin P. ; Donaldson, Paul M. ; Greetham, Gregory M. ; Towrie, Michael ; Parker, Anthony W. ; Baker, Matthew J. ; Henry, Alistair J. ; Taylor, Richard J. ; Hunt, Neil T./ Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy. In: Analytical Chemistry. 2017 ; Vol. 89, No. 20. pp. 10898-10906
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abstract = "Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13{\%} (CD) and 15{\%} (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally-stable Ca2+-bound (holo-) CaM. The functionally-relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.",
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author = "Lucy Minnes and Shaw, {Daniel J.} and Cossins, {Benjamin P.} and Donaldson, {Paul M.} and Greetham, {Gregory M.} and Michael Towrie and Parker, {Anthony W.} and Baker, {Matthew J.} and Henry, {Alistair J.} and Taylor, {Richard J.} and Hunt, {Neil T.}",
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Minnes, L, Shaw, DJ, Cossins, BP, Donaldson, PM, Greetham, GM, Towrie, M, Parker, AW, Baker, MJ, Henry, AJ, Taylor, RJ & Hunt, NT 2017, 'Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy' Analytical Chemistry, vol. 89, no. 20, pp. 10898-10906. DOI: 10.1021/acs.analchem.7b02610

Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy. / Minnes, Lucy; Shaw, Daniel J.; Cossins, Benjamin P.; Donaldson, Paul M.; Greetham, Gregory M.; Towrie, Michael; Parker, Anthony W.; Baker, Matthew J.; Henry, Alistair J.; Taylor, Richard J.; Hunt, Neil T.

In: Analytical Chemistry, Vol. 89, No. 20, 17.10.2017, p. 10898-10906.

Research output: Contribution to journalArticle

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T1 - Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy

AU - Minnes,Lucy

AU - Shaw,Daniel J.

AU - Cossins,Benjamin P.

AU - Donaldson,Paul M.

AU - Greetham,Gregory M.

AU - Towrie,Michael

AU - Parker,Anthony W.

AU - Baker,Matthew J.

AU - Henry,Alistair J.

AU - Taylor,Richard J.

AU - Hunt,Neil T.

N1 - This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.7b02610.

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Y1 - 2017/10/17

N2 - Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally-stable Ca2+-bound (holo-) CaM. The functionally-relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

AB - Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally-stable Ca2+-bound (holo-) CaM. The functionally-relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

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KW - data analysis

KW - structure

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Minnes L, Shaw DJ, Cossins BP, Donaldson PM, Greetham GM, Towrie M et al. Quantifying secondary structure changes in Calmodulin using 2D-IR spectroscopy. Analytical Chemistry. 2017 Oct 17;89(20):10898-10906. Available from, DOI: 10.1021/acs.analchem.7b02610