Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY
Public Deposited- Last Modified
- March 22, 2019
- Creator
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McDonald, Leanna Rose
- Affiliation: School of Medicine, Department of Biochemistry and Biophysics, Gillings School of Global Public Health
- Abstract
- Allosteric proteins are extremely important in signaling, yet the mechanism(s) of the switch between inactive and active conformations is not clearly understood. It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. By utilizing 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that unphosphorylated CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the ms-ms timescale motions underlying CheY allostery are segmental in nature. Upon phosphorylation, CheY allosterically responds with a change in dynamics on both the μs-ms and ps-ns timescale. We observe an apparent decrease and redistribution of μs-ms dynamics upon phosphorylation of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by μs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the μs-ms and ps-ns timescales in CheY. Analysis of an activating mutant A113P yields a similar pattern of side-chain ps-ns dynamic changes as upon phosphorylation. This relationship between an activating mutant and an activating pattern of dynamic changes is further evidence for a distinct relationship between the dynamics on multiple timescales and the function of the protein. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function and the complex mechanisms proteins utilize for allostery.
- Date of publication
- August 2013
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- Rights statement
- In Copyright
- Advisor
- Lee, Andrew
- Degree
- Doctor of Philosophy
- Graduation year
- 2013
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