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

TY - JOUR

T1 - Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy

AU - Minnes, Lucy

AU - Shaw, Daniel J

AU - Cossins, Benjamin P

AU - Donaldson, Paul M

AU - Greetham, Gregory M

AU - Towrie, Michael

AU - Parker, Anthony W

AU - Baker, Matthew J

AU - Henry, Alistair J

AU - Taylor, Richard J

AU - Hunt, Neil T

PY - 2017/10/17

Y1 - 2017/10/17

N2 - (Graph Presented) 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.

AB - (Graph Presented) 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.

UR - http://www.scopus.com/inward/record.url?scp=85031400594&partnerID=8YFLogxK

U2 - 10.1021/acs.analchem.7b02610

DO - 10.1021/acs.analchem.7b02610

M3 - Article

C2 - 28921967

SN - 0003-2700

VL - 89

SP - 10898

EP - 10906

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 20

ER -