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

5 years ago

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