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Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
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At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
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Many organic, inorganic, and biological molecules contain spin-half nuclei such as nitrogen-15, fluorine-19, and phosphorus-31. As a result, NMR studies of these nuclei have found extensive applications in chemical and biological research.
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Organic molecules primarily contain carbon and hydrogen atoms. While all the hydrogen isotopes are NMR-active, protium or hydrogen-1 is the most abundant. It has a significant energy separation between its nuclear spin states due to its large gyromagnetic ratio. As per Boltzmann's distribution, an increase in the energy separation implies a greater excess population of nuclei available for excitation, resulting in a strong NMR absorption signal.
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Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

Venkatraman Ramanujam1, T Reid Alderson1, Iva Pritišanac1

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt am Main, 60438 Frankfurt am Main, Germany.

Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|March 1, 2020
PubMed
Summary

High pressure alters the hydrodynamic radius (Rh) of small molecules and unfolded proteins by increasing their coupling with water. This effect is concentration-dependent for biomolecules like ubiquitin and α-synuclein.

Keywords:
DioxaneHydration layerIDPPressure-induced unfoldingProtein compactionRadius of hydrationRandom coilSelf-diffusionTranslational diffusionUbiquitinUnfolded chainUreaα-synuclein

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Area of Science:

  • Biophysical Chemistry
  • Protein Dynamics
  • Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

  • Pulsed-field gradient NMR spectroscopy measures translational diffusion and hydrodynamic radius (Rh) of biomolecules.
  • Rh of unfolded proteins reports on conformation and chain expansion under denaturant.
  • Hydrostatic pressure is a reversible alternative to chemical denaturants for protein folding studies.

Purpose of the Study:

  • To investigate the effect of hydrostatic pressure on the hydrodynamic radius (Rh) and translational diffusion of small molecules and unfolded proteins.
  • To compare pressure-induced denaturation with urea-induced denaturation.
  • To assess the influence of sample concentration on diffusion measurements.

Main Methods:

  • Pulsed-field gradient NMR spectroscopy to measure translational diffusion.
  • Hydrodynamic radius (Rh) determination from diffusion coefficients.
  • 13C longitudinal relaxation times to assess rotational friction.
  • Measurements performed as a function of hydrostatic pressure and urea concentration.

Main Results:

  • Elevated pressures increase Rh of small molecules (e.g., dioxane) correlating with hydrophobicity, indicating tighter hydrophobic-water coupling.
  • Rh of folded and unfolded states of a ubiquitin mutant (VA2-ubiquitin) remained invariant with pressure or urea.
  • Pressure-denatured ubiquitin Rh (ca 23 Å) was indistinguishable from urea-denatured state and an idealized random coil.
  • Intrinsically disordered protein (IDP) α-synuclein showed slight compaction above 2 kbar.
  • Diffusion of unfolded ubiquitin and α-synuclein was concentration-dependent.

Conclusions:

  • Hydrostatic pressure enhances the interaction between hydrophobic surfaces and water, affecting Rh of small molecules.
  • Pressure-induced unfolding of ubiquitin results in an Rh similar to urea-induced unfolding and random coil models.
  • Accurate diffusion measurements of unfolded proteins require dilute conditions due to concentration effects.