Title
Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies
Author
Huma Naz, Mohd. Shahbaaz, Krishna Bisetty, Asimul Islam, Faizan Ahmad, Imtaiyaz Hassan
Year
2016
Journal
Biochemistry and Cell Biology
Abstract
Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium–calmodulin dependent signal through a secondary messenger, Ca2+, which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca2+ concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0–11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0–4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV.
Instrument
FP-6200
Keywords
Fluorescence, Protein structure, Chemical stability, Biochemistry