3 years ago

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy
Matthew J. Baker, Benjamin P. Cossins, Michael Towrie, Neil T. Hunt, Paul M. Donaldson, Gregory M. Greetham, Anthony W. Parker, Daniel J. Shaw, Richard J. Taylor, Alistair J. Henry, Lucy Minnes
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.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b02610

DOI: 10.1021/acs.analchem.7b02610

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