Thermal perturbation correlation of calcium binding Human centrin 3 and its structural changes

•PCMW2D correlation spectroscopy has proven useful in defining Tm of protein motifs.•The aspartates and glutamates vibrational modes were analyzed.•The N-terminal domain has high affinity for calcium and therefore high Tm.•The sequential orders of events were validated.

Perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy was applied for the determination of the individual transition temperatures of different vibrational modes located within structural components of a calcium binding protein known as Human centrin 3. This crucial information served to understand the contribution individual calcium binding sites made towards the stability of the EF-hand and therefore the protein without the use of probes. We are convinced that the general application of PCMW2D correlation spectroscopy can be applied to the study of proteins in general to ascertain the differences in the stability of structural motifs within proteins and its relationship to the actual transition temperature of unfolding.