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

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(Graph Presented) 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 Ca 2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca 2+ -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 Ca 2+ -bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca 2+ 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 Ca 2+ 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.

Original languageEnglish
Pages (from-to)10898-10906
Number of pages9
JournalAnalytical Chemistry
Issue number20
Early online date29 Sept 2017
Publication statusPublished - 17 Oct 2017

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