Colocalization of Fast and Slow Timescale Dynamics in the Allosteric Signaling Protein CheY

• CheY switches between inactive and active-like conformations on the microsecond-to-millisecond timescale.
• Phosphorylation dampens and redistributes microsecond-to-millisecond motions in CheY.
• Phosphorylation induces changes in picosecond-to-nanosecond side-chain dynamics in CheY.
• We observe limited global spreading of picosecond-to-nanosecond dynamic changes in CheY.
• Picosecond-to-nanosecond dynamic changes localize to the allosteric conformational change region.

It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. 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. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the microsecond-to-millisecond (μs-ms) timescale and the picosecond-to-nanosecond (ps-ns) timescale. We observe an apparent decrease and redistribution of μs-ms dynamics upon phosphorylation (and accompanying Mg2 + saturation) 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. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.

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