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Related Concept Videos

Atomic Nuclei: Nuclear Spin State Overview01:03

Atomic Nuclei: Nuclear Spin State Overview

NMR-active nuclei have energy levels called 'spin states' that are associated with the orientations of their nuclear magnetic moments. In the absence of a magnetic field, the nuclear magnetic moments are randomly oriented, and the spin states are degenerate. When an external magnetic field is applied, the spin states have only 2 + 1 orientations available to them. A proton with = ½ has two available orientations. Similarly, for a quadrupolar nucleus with a nuclear spin value of one, the...
Atomic Nuclei: Nuclear Spin01:08

Atomic Nuclei: Nuclear Spin

All atomic particles possess an intrinsic angular momentum, or 'spin'. Electrons, protons, and neutrons each have a spin value of ½, although protons and neutrons in nuclei may have higher half-integer spins owing to energetic factors.
Atomic nuclei have a net nuclear spin, , which can have an integer or half-integer value. In atomic nuclei, the spins of protons are paired against each other but not with neutrons, and vice versa. Consequently, an even number of protons does not contribute to...
¹H NMR: Interpreting Distorted and Overlapping Signals01:02

¹H NMR: Interpreting Distorted and Overlapping Signals

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.
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...
NMR Spectroscopy: Spin–Spin Coupling01:08

NMR Spectroscopy: Spin–Spin Coupling

The spin state of an NMR-active nucleus can have a slight effect on its immediate electronic environment. This effect propagates through the intervening bonds and affects the electronic environments of NMR-active nuclei up to three bonds away; occasionally, even farther. This phenomenon is called spin–spin coupling or J-coupling. Coupling interactions are mutual and result in small changes in the absorption frequencies of both nuclei involved. While nuclei of the same element are involved in...
Atomic Nuclei: Nuclear Magnetic Moment00:59

Atomic Nuclei: Nuclear Magnetic Moment

All atomic nuclei are positively charged. When they have a nonzero spin, they behave like rotating charges. As a consequence of their charge and spin, these nuclei generate a magnetic field (B). This, in turn, gives rise to a magnetic moment (μ), which is randomly oriented in the absence of an external magnetic field. When an external magnetic field (B0) is applied, the magnetic moment vectors can align with the field or against it in 2 + 1 orientations. A hydrogen nucleus, which is just a...
Atomic Nuclei: Nuclear Relaxation Processes01:23

Atomic Nuclei: Nuclear Relaxation Processes

In the absence of an external magnetic field, nuclear spin states are degenerate and randomly oriented. When a magnetic field is applied, the spins begin to precess and orient themselves along (lower energy) or against (higher energy) the direction of the field. At equilibrium, a slight excess population of spins exists in the lower energy state. Because the direction of the magnetic field is fixed as the z-axis,  the precessing magnetic moments are randomly oriented around the z-axis. This...

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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
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Structural origin of weakly ordered nitroxide motion in spin-labeled proteins.

Mark R Fleissner1, Duilio Cascio, Wayne L Hubbell

  • 1Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008.

Protein Science : a Publication of the Protein Society
|April 23, 2009
PubMed
Summary

Site-directed spin labeling with R1 nitroxide probes reveals a common disulfide-backbone interaction governing internal motion. This interaction explains observed anisotropic motion across diverse protein sites, attributing variations to backbone dynamics.

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Area of Science:

  • Biophysics
  • Structural Biology
  • Protein Dynamics

Background:

  • Site-directed spin labeling (SDSL) using disulfide-linked nitroxide side chains (R1) is a key technique in protein studies.
  • R1 probes often show EPR spectra indicative of weakly ordered z-axis anisotropic motion at various protein surface sites.

Purpose of the Study:

  • To determine the structural basis for the observed anisotropic motion of R1 probes at different protein locations.
  • To investigate the role of specific interactions in R1 side chain dynamics.

Main Methods:

  • X-ray crystallography was used to determine the structures of T4 lysozyme with R1 probes at helical (131, 151) and loop (82) sites.
  • Electron Paramagnetic Resonance (EPR) spectroscopy was employed to analyze the motion of the R1 probes.

Main Results:

  • Crystal structures revealed an intraresidue C(alpha)--H...S(delta) interaction immobilizing the disulfide group in helical sites.
  • This disulfide-backbone interaction was also observed at a loop site, indicating it is not dependent on secondary structure.
  • Despite populating two rotamers, R1 at site 131 exhibited a single dominant EPR dynamic population due to similar internal motion.

Conclusions:

  • The internal motion of R1 probes is primarily governed by rotations around terminal bonds, influenced by a common disulfide-backbone interaction.
  • This interaction is consistent across helical and loop sites, suggesting it's a general feature.
  • Observed variations in R1 dynamics at solvent-exposed sites are likely attributable to protein backbone motion, as R1's internal motion remains relatively constant.