Two-dimensional infrared correlation analysis of protein unfolding: Use ofspectral simulations to validate structural changes during thermal denaturation of bacterial CMP kinases

The functional role of bacterial CMP kinases is to recover the energeticall y exhausted nucleoside monophosphates derived from cell metabolism by trans ferring a phosphate residue from ATP to CMP or dCMP. These enzymes-importan t for cell growth and division-possess two distinct binding sites and a num ber of conserved secondary structure elements. Herein we compare the infrar ed spectra of two similar, but not identical, CMP kinases from Escherichia coli and Bacillus subtilis. The two-dimensional correlation analysis of the infrared spectra of the two enzymes reveals significant differences in pro tein structure upon denaturation, a fact possibly linked to their different biochemical and catalytic properties. Model calculations are used to illus trate the effect of two separate processes on the out-of-phase correlation in the two-dimensional (2D) correlation plots. This strategy is then employ ed to validate the changes observed in the secondary structure of the two e nzymes. When bound to the active site of the protein, the two substrates CM P and ATP exert a stabilizing effect on the structure of both proteins; how ever, the changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations that simulate the denaturation of the tw o enzymes confirm the occurrence of temperature-delayed unfolding processes in both proteins. Thermal denaturation and aggregation can be distinguishe d in both proteins as two distinct processes, separated in time.