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Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

ABSTRACT: Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (R_h) of biomolecules in solution. For unfolded proteins, the R_h provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. While the impact of pressure on the viscosity of water is well known, and our water diffusivity measurements agree closely with theoretical expectations, we find that elevated pressures increase the R_h of dioxane and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction indicated by ¹³C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurement of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure or urea concentration shows that R_h values of both the folded and the unfolded states remain nearly invariant. At ca 23 Å, the R_h of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) ?-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of unfolded ubiquitin and ?-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions.

SUBMITTER: Ramanujam V

PROVIDER: S-EPMC7153785 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

Ramanujam Venkatraman V Alderson T Reid TR Pritišanac Iva I Ying Jinfa J Bax Ad A

Journal of magnetic resonance (San Diego, Calif. : 1997) 20200219

Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (R<sub>h</sub>) of biomolecules in solution. For unfolded proteins, the R<sub>h</sub> provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measuremen ...[more]

PMID: 32113145

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Project description:Monitoring time dependence with diffusion MRI yields observables sensitive to compartment sizes (restricted diffusion) and membrane permeability (water exchange). However, restricted diffusion and exchange have opposite effects on the diffusion-weighted signal, which can lead to errors in parameter estimates. In this work, we propose a signal representation that incorporates the effects of both restricted diffusion and exchange up to second order in b-value and is compatible with gradient waveforms of arbitrary shape. The representation features mappings from a gradient waveform to two scalars that separately control the sensitivity to restriction and exchange. We demonstrate that these scalars span a two-dimensional space that can be used to choose waveforms that selectively probe restricted diffusion or exchange, eliminating the correlation between the two phenomena. We found that waveforms with specific but unconventional shapes provide an advantage over conventional pulsed and oscillating gradient acquisitions. We also show that parametrization of waveforms into a two-dimensional space can be used to understand protocols from other approaches that probe restricted diffusion and exchange. For example, we found that the variation of mixing time in filter-exchange imaging corresponds to variation of our exchange-weighting scalar at a fixed value of the restriction-weighting scalar. The proposed signal representation was evaluated using Monte Carlo simulations in identical parallel cylinders with hexagonal and random packing as well as parallel cylinders with gamma-distributed radii. Results showed that the approach is sensitive to sizes in the interval 4-12 μm and exchange rates in the simulated range of 0 to 20 s-1 , but also that there is a sensitivity to the extracellular geometry. The presented theory constitutes a simple and intuitive description of how restricted diffusion and exchange influence the signal as well as a guide to protocol design capable of separating the two effects.

Dataset Information

Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

Publications

Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

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