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

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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.