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¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

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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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¹H NMR: Interpreting Distorted and Overlapping Signals01:02

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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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NMR Spectroscopy: Spin–Spin Coupling01:08

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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...
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NMR Spectrometers: Resolution and Error Correction01:14

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When magnetic nuclei in a sample achieve resonance and undergo relaxation, the signal detected in NMR is an approximately exponential free induction decay. Fourier transform of an exponential decay yields a Lorentzian peak in the frequency domain. Lorentzian peaks in an NMR spectrum are defined by their amplitude, full width at half maximum, and position, where the peak width is governed by the spin-spin relaxation time alone. In real experiments, however, the applied magnetic field is rendered...
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Two-Dimensional (2D) NMR: Overview01:12

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The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
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¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

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The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
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Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments
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Enzyme dynamics from NMR spectroscopy.

Arthur G Palmer1

  • 1Department of Biochemistry and Molecular Biophysics, Columbia University , 701 West 168th Street, New York, New York 10032, United States.

Accounts of Chemical Research
|January 10, 2015
PubMed
Summary
This summary is machine-generated.

Enzyme function relies on conformational changes. NMR spin relaxation reveals how protein flexibility and dynamics influence enzyme mechanisms, from substrate binding to product release, offering insights into enzyme regulation and activity.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Enzyme activity is modulated by conformational dynamics, affecting key steps like substrate binding and product release.
  • Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for studying these dynamic processes at atomic resolution across various timescales.

Purpose of the Study:

  • To review NMR spin relaxation studies investigating the role of conformational flexibility and dynamics in enzyme mechanisms.
  • To illustrate these principles using examples from ribonuclease HI, AlkB, and triosephosphate isomerase.

Main Methods:

  • NMR spin relaxation measurements (laboratory-frame and rotating-frame relaxation-dispersion) to probe motions on picosecond-nanosecond and microsecond-millisecond timescales.
  • Molecular dynamics (MD) simulations to complement experimental data and provide atomic-level insights.
  • Fluorescence spectroscopy for AlkB conformational studies.

Main Results:

  • Ribonuclease H: Interconversion between two conformations in the handle region, with the closed state potentially inhibiting substrate binding and contributing to higher Michaelis constants in thermophilic variants.
  • AlkB: A conformational transition between open and closed states, regulated by cosubstrate 2-oxoglutarate (2OG), ensuring proper substrate binding order and preventing premature substrate release.
  • Triosephosphate isomerase: Loop 6 closure is crucial for Michaelis complex formation, while its reopening is rate-limiting for product release, involving correlated rigid-body motion and specific residue dynamics.

Conclusions:

  • NMR spin relaxation, combined with MD simulations, provides comprehensive insights into how enzyme conformational dynamics govern catalytic mechanisms.
  • These dynamic processes are critical for enzyme regulation, substrate recognition, and efficient catalysis, from initial activation to product release.