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

Lucy Minnes

doi:10.1021/acs.analchem.7b02610

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

2017-09-29

36

OA

AI

DOI：10.1021/acs.analchem.7b02610

Original

Save

View PDF

Abstract

En 中文

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 alpha-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-alpha-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.

Keywords:

2-DIMENSIONAL INFRARED-SPECTROSCOPY

2D IR SPECTROSCOPY

CIRCULAR-DICHROISM SPECTROSCOPY

ISLET AMYLOID POLYPEPTIDE

CALCIUM-BINDING

APO-CALMODULIN

TROPONIN-C

CONFORMATIONAL-CHANGES

QUANTITATIVE-ANALYSIS

RAMAN-SPECTROSCOPY

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

Abstract

AI Summary

Journal

Organization