Two-Dimensional Infrared Correlation Analysis of Protein Unfolding: Use of Spectral Simulations to Validate Structural Changes during Thermal Denaturation of Bacterial CMP Kinases
| Autor: | Christian P. Schultz, Octavian Bârzu, Henry H. Mantsch |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Bacillus subtilis
01 natural sciences 010309 optics Protein structure Protein secondary structure analysis 2D-IR 0103 physical sciences Denaturation (biochemistry) Protein folding Binding site Instrumentation Protein secondary structure Spectroscopy chemistry.chemical_classification biology Chemistry 010401 analytical chemistry Active site biology.organism_classification 0104 chemical sciences Enzyme Biochemistry FT-IR spectroscopy biology.protein Two-dimensional IR correlation spectroscopy |
| Zdroj: | Applied Spectroscopy. 54:931-938 |
| ISSN: | 1943-3530 0003-7028 |
| DOI: | 10.1366/0003702001950562 |
| Popis: | The functional role of bacterial CMP kinases is to recover the energetically exhausted nucleoside monophosphates derived from cell metabolism by transferring a phosphate residue from ATP to CMP or dCMP. These enzymes—important for cell growth and division—possess two distinct binding sites and a number of conserved secondary structure elements. Herein we compare the infrared 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 protein structure upon denaturation, a fact possibly linked to their different biochemical and catalytic properties. Model calculations are used to illustrate the effect of two separate processes on the out-of-phase correlation in the two-dimensional (2D) correlation plots. This strategy is then employed to validate the changes observed in the secondary structure of the two enzymes. When bound to the active site of the protein, the two substrates CMP and ATP exert a stabilizing effect on the structure of both proteins; however, the changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations that simulate the denaturation of the two enzymes confirm the occurrence of temperature-delayed unfolding processes in both proteins. Thermal denaturation and aggregation can be distinguished in both proteins as two distinct processes, separated in time. |
| Databáze: | OpenAIRE |
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