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

Abstract

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 concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca-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-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca 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 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.

Measuring proteins in H2O with 2D-IR spectroscopy

“…In principle, 2D-IR offers the scope to go beyond the AGR measurement by virtue of the information-rich 2D lineshape of the proteins, which gives the opportunity to resolve more than albumin and the γ-globulins 4. The major protein constituent of the γ-globulins is IgG (80%).…”

Biofluid analysis and classification using IR and 2D-IR spectroscopy

“…2D-IR spectroscopy 2D-IR spectroscopy has been used to probe the molecular vibrations of many systems including biological molecules and solvents and has provided visualisations of conformational structural changes and dynamics of molecules [25,29,[112][113][114][115][116][117][118][119][120]. Proof of principle studies have shown the power and sensitivity of 2D-IR as it observes molecular structures and changes within a system allowing identification of proteins, ligands, DNA and other markers, and it has the potential to aid in disease diagnosis [11,29,113,116,[120][121][122][123][124][125]. However the use of 2D-IR to study biofluids is in its infancy and requires development which faces many challenges.…”

“…PCA has been shown to be a powerful tool for use with 2D-IR spectroscopy and has enabled extraction of information from 2D-IR datasets [23,29,122]. It is important to note that prior to fit using a bilinear model such as PCA, the data must be first altered into a suitable format, ideally where each 2D matrix is reshaped into a single dimensional vector.…”

Two-dimensional infrared spectroscopy: an emerging analytical tool?

“…1, 4-11 As drug design turns towards therapeutic molecules that target dynamic protein structures, such time resolved insight may become a powerful tool. 26 In such cases, the ability to measure changes in structure, often affecting a small fraction of the overall protein Please do not adjust margins Please do not adjust margins molecule, may become important. With this in mind, several 2D-IR studies have assessed the ability to identify, categorise and measure protein structures.…”

“…It is thus important that 2D-IR has been shown to provide similar accuracy in terms of quantification of secondary structure, with a study of the Ca 2+ binding transition of calmodulin showing that 2D-IR spectroscopy is sensitive to a secondary structure change affecting just seven residues of the 150 residue protein. 26 Thus far, we have focused on 2D-IR experiments using broadband, short duration infrared pulses as the source of the excitation and detection pulses. However, a second, less widely used 2D-IR method called electron-vibration-vibration (EVV) 2D-IR has been applied to protein structure determination.…”