Abstract
Numerous studies have suggested a significant role that protein dynamics play in optimizing enzyme catalysis, and changes in conformational sampling offer a window to explore this role. Thermolysin from Bacillus thermoproteolyticus rokko, which is a heat-stable zinc metalloproteinase, serves here as a model system to study changes of protein function and conformational sampling across a temperature range of 16–36 °C. The temperature dependence of kinetics of thermolysin showed a biphasic transition at 26 °C that points to potential conformational and dynamic differences across this temperature. The non-Arrhenius behavior observed resembled results from previous studies of a thermophilic alcohol dehydrogenase enzyme, which also indicated a biphasic transition at ambient temperatures. To explore the non-Arrhenius behavior of thermolysin, room temperature crystallography was applied to characterize structural changes in a temperature range across the biphasic transition temperature. The alternate conformation of side chain fitting to electron density of a group of residues showed a higher variability in the temperature range from 26 to 29 °C, which indicated a change in conformational sampling that correlated with the non-Arrhenius break point.
Highlights
The alternate conformation of side chain fitting to electron density of a group of residues showed a higher variability in the temperature range from 26 to 29 C, which indicated a change in conformational sampling that correlated with the non-Arrhenius break point
There have been studies utilizing hydrogen/ deuterium exchange (HDX) mass spectrometry that support a direct correlation between the time scale of conformational fluctuations and the turnover number of the enzyme thermolysin by showing that the substrate turnover is associated with the hinge bending that leads to a closed conformation
Our current results strongly suggest a dynamic biphasic transition in protein conformational sampling that has improved catalytic efficiency above this temperature
Summary
Protein conformational changes are essential for enzyme function, and increased protein conformational flexibility has been linked to enhanced enzymatic activity. Protein flexibility has been described by conformational disorder, observed by crystallography or nuclear magnetic resonance (NMR), and by hydrogen/ deuterium exchange (HDX) approaches. Notably, there have been studies utilizing HDX mass spectrometry that support a direct correlation between the time scale of conformational fluctuations and the turnover number of the enzyme thermolysin by showing that the substrate turnover is associated with the hinge bending that leads to a closed conformation.11temperature has been used as a probe to study protein dynamics, and changes to structural stability and conformational dynamics of proteins have shown interesting results. Several enzyme systems have suggested that there is a biphasic transition in protein conformational sampling. Protein conformational changes are essential for enzyme function, and increased protein conformational flexibility has been linked to enhanced enzymatic activity.. Protein flexibility has been described by conformational disorder, observed by crystallography or nuclear magnetic resonance (NMR), and by hydrogen/ deuterium exchange (HDX) approaches.. Temperature has been used as a probe to study protein dynamics, and changes to structural stability and conformational dynamics of proteins have shown interesting results.. Several enzyme systems have suggested that there is a biphasic transition in protein conformational sampling. Biphasic conformational dynamics behavior was observed from À53 to À23 C in Zn-substituted cytochrome c peroxidase by studying the quenching of the 3ZnP excited state.. In myoglobin and ribonuclease A enzyme systems, ligands were shown to bind to the protein only above a critical and specific temperature: À93 C for myoglobin and À53 C for ribonuclease A.13,16. Myoglobin showed extra mobility above À93 C from neutron scattering results. Biphasic conformational dynamics behavior was observed from À53 to À23 C in Zn-substituted cytochrome c peroxidase by studying the quenching of the 3ZnP excited state. In myoglobin and ribonuclease A enzyme systems, ligands were shown to bind to the protein only above a critical and specific temperature: À93 C for myoglobin and À53 C for ribonuclease A.13,16 In ribonuclease A, there was a biphasic break in the temperature dependence of the thermal B factor, indicating a biphasic protein dynamic behavior from À93 to À73 C.9 Turning attention to a thermophilic enzyme, the alcohol dehydrogenase from
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