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

Research output: Research - peer-reviewArticle

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.
Original languageEnglish
Pages (from-to)10898-10906
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number20
Early online date18 Sep 2017
DOIs
StatePublished - 17 Oct 2017

    Research areas

  • spectroscopy, data analysis, structure, Calmodulin

Bibliographical note

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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