Changes in Secondary Structure of Key Calcium Binding Protein Quantified at CLF
15 Nov 2017
- Emily Cooke



Scientists from the University of Strathclyde, UCB Pharma and STFC’s Central Laser Facility have used 2D-IR spectroscopy to quantify changes in the secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM).




Making use of the ULTRA laser facility at the Central Laser Facility (CLF), the research team has been able to measure subtle protein conformational changes in solution - as a function of temperature and Calcium (Ca2+) concentration - using 2D-IR spectroscopy in combination with multivariate analysis. This work opens up the possibility to observe structural changes of proteins in solution in real time via a label-free method.

CLF's Ultra laser, housed within the Research Complex at Harwell, is one of the world's most sensitive 2D-IR spectrometers and is used to investigate dynamics of complex biological systems such as the protein Calmodulin studied in this experiment.  

By measuring the thermal stability and Ca2+ - binding transition of human CaM with IR absorption and 2D-IR spectroscopy, the team have proved that 2D-IR can be used to reliably quantify relative changes in secondary structure elements in proteins by measuring subtle changes in the 2D-IR spectra of the amide I IR absorption band. Not only can ultrafast 2D-IR spectroscopy of unlabelled proteins help scientists paint a broader picture of conformational change but it can also help create a platform for spatially targeted studies.

The principle of the 2DIR technique is to use ultrashort pulse of IR laser light to look at (probe) the response of molecules after vibrations within them have been excited (pumped) by pulses of IR light. A 2DIR spectrum shows the change in the probe spectrum as a function of pump wavelength and provides information on how vibrations couple to other vibrations within the protein secondary structure as well as how excited vibrations lose their energy. This reports on structure but also on fast motions or chemical changes occurring.  The technique requires ultrashort (femtosecond) IR pulsed light because in the case of proteins they “forget" that they have been pumped after only a few picoseconds and they must be probed before this happens.    

The protein in question, Calmodulin is a universal, multifunctional Ca2+ binding protein, responsible for many of the regulatory effects of calcium which include smooth muscle contraction, intracellular signalling and cell cycle regulation. In smooth muscle, Ca2+ binds to calmodulin and allows it to activate an enzyme called myosin light chain kinase which catalyses the phosphorylation of the motor protein myosin. The result of this cascade of intracellular activity is smooth muscle contraction. As structure and function in biological molecules are so heavily linked, an understanding of the molecular mechanisms that occur under physiological conditions must be quantified.

Data from the paper [1], published in the Journal Analytical Chemistry indicates that secondary structure transitions, along with thermally induced alterations in the coupling between vibrations in the protein, alters the 2D-IR signal.  It is also evident from the results that the signal from 2D-IR is more sensitive to thermally induced 'relaxation' of coupling within the α -helix than conventional IR spectroscopy  and that it complements UV Circular Dichroism Spectroscopy often used in these types of measurements. Furthermore, the results reveal that 2D-IR spectroscopy is sensitive to changes in secondary structure at the few percent level, which means that it provides superior contrast to IR absorption when considering Ca2+ induced structural change in human CaM.

By revealing quantitative structural insight into Calmodulin, Minnes et al have proven that ultrafast 2D-IR -in combination with Principal Component Analysis - can act as an accurate, efficient reporter of protein secondary structure change. This makes it very useful for characterising complex systems and increasing our understanding of the molecular mechanisms involved. 

The research was supported by STFC and the full publication is available to view in the journal Analytical Chemistry.

For further information about the research, please contact Prof Neil Hunt (

To read more news releases concerning the CLF, please visit CLF News .

[1] L. Minnes, D. J. Shaw, B. P. Cossins, P. A. Donaldson, G. M. Greetham, M. Towrie, A. W. Parker, M. J. Baker, A. J. Henry, R. J. Taylor and N. T. Hunt, "Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy," Analytical Chemistry , pp. 10898-10906, 2017. 


Contact: Cooke, Emily (STFC,RAL,ISIS)