Exploring cavity effects on protein dynamic disorder with pressure perturbation

Zhang, Siwen
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Wang, Chunyu
Makhatadze, George I.
Colón, Wilfredo
Royer, Catherine Ann
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Given the central role of conformational dynamics in protein function, it is essential to characterize the timescales and structures associated with these transitions. High-pressure perturbation favors transitions to excited states because they typically occupy a smaller molar volume, thus high pressure facilitates the characterization of conformational dynamics. In this dissertation, we describe the use of a combination of NMR chemical exchange spectroscopy, small-angle X-ray scattering, and high hydrostatic pressure to better investigate conformational exchange during protein folding process. Repeat proteins, with their straightforward architecture, provide good models for probing the sequence dependence of protein conformational dynamics. We choose the leucine rich repeat (LRR) domain of the tumor suppressor pp32 as a model. Pp32 is composed of five LRRs with a capping motif on each of its termini. We show here that the introduction of a cavity in the N-terminal capping motif of pp32 leads to pressure-dependent conformational exchange detected on the 500 µs - 2 ms timescale by 15N CPMG relaxation dispersion analysis. Exchange amplitude and minimum chemical shifts decrease from the N- to the C-terminus, revealing a gradient of structural disruption across the protein. In contrast, introduction of a cavity in the central core of pp32 leads to pressure-induced exchange on a slower (> 2 ms) timescale detected by 15N-CEST analysis. Excited state 15N chemical shifts indicate that in the major excited state, the N-terminal region is mostly unfolded, while the core retains native-like structure. These high-pressure chemical state exchange measurements reveal that cavity position dictates distinct structural dynamics, highlighting the subtle, yet central role of sequence in determining protein conformational dynamics.
August 2022
School of Science
Dept. of Chemistry and Chemical Biology
Rensselaer Polytechnic Institute, Troy, NY
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