Studies Of The Domain-domain Interactions And Polar Microenvironment Around The Active Sites Of Creatine Kinase

Many large proteins are composed of domains that can be regarded as structure, function, evolution and folding units. For these multidomain proteins, folding is generally thought to be hierarchical and the individual domains with different stabilities fold autonomously and independently. However, the folding mechanism of multidomain proteins may be much more complicated than single-domain proteins because it involves both the folding of the individual domains and the docking of these domains. Domain folding and assembly play a role in the stability of the domains or the whole protein, even for their functions in vivo, when domain-domain interactions is present. The domains are connected sequentially by the linker in the primary structure. However, little is known about the role of the linker in protein folding, stability and function.The aggregation occurred at a temperature (about 47℃) far below the Tm (about 56℃) of creatine kinase. However, the mechanism of the fast destabilization and aggregation of creatine kinase induced by heat is not clear yet. In this research, the sequential events of creatine kinase thermal inactivation and aggregation were studied by quantitative second derivative infrared analysis and two-dimensional infrared correlation spectroscopy, combined with circular dichroism spectroscopy and tryptophan fluorescense spectroscopy. The results indicated that C-terminal domain played an important role in the structural stability and folding of creatine kinase. We presented a possible mechanism of creatine kinase thermal inactivation and aggregation and suggested that the conformational change in the C-terminal domain was responsible for the initiation of creatine kinase thermal aggregation.The domains are connected sequentially by the linker in the primary structure. The linker does not form any regular secondary structures and seems to be flexible. Moreover, the linker is mainly composed of charged residues and only contains four hydrophobic conserved residues. In this research, the results suggested that the hydrophobic interactions between the linker and the C-terminal domain played a key role on the activity, structural stability and folding of creatine kinase by mutation and spectral analysis. The location of the linker sequence may be helpful to the natvie, active conformation formation assemble by the two domains of creatine kinase correctly.The polarities of protein have an important role in the structures, catalysis and functions. In this research, 5,5 -?dithiobis(2-nitrobenzoic acid), 6,6 -?dithio- dinicotinic acid and 2,2 -?dithiodipyridine were used as specific sulphydryl reagents to explore the polar microenvironment around Cys283, a conserved amino acid nearby the active site of creatine kinase. Complete kinetics of inactivation was recorded, and the relevant microscopic rate constants of the reagents with various enzyme-substrate complexes were also determined. The results indicated that there may exist a polar microenvironment which is predominant by the electropositive residues and this may be the reason for the unusually low pKa value of Cys283.