Characterization of lanthanoid-binding proteins using NMR spectroscopy
Enrico Ravera1, Linda Cerofolini2, Marco Fragai1
1Magnetic Resonance Center (CERM) and Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, Italy; Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (CIRMMP), Sesto Fiorentino, Italy.
Methods in Enzymology
|April 23, 2021
Summary
Paramagnetic lanthanoids offer valuable insights into protein structures using Nuclear Magnetic Resonance (NMR) spectroscopy. This chapter details their application in structural biology, from sample preparation to data analysis for characterizing lanthanoid-binding proteins.
Area of Science:
- Structural biology
- Biophysical chemistry
- Nuclear Magnetic Resonance (NMR) spectroscopy
Background:
- Paramagnetic lanthanoids possess unique magnetic properties beneficial for structural biology.
- These properties enable the characterization of lanthanoid-binding proteins.
- Paramagnetic restraints, including paramagnetic chemical shift (PCS), paramagnetic relaxation (PRDC), and paramagnetic relaxation enhancement (PRE), offer insights into protein structure and dynamics.
Purpose of the Study:
- To elucidate the utility of lanthanoids as a tool in NMR-based structural biology.
- To provide a comprehensive overview of lanthanoid applications in protein structure determination.
- To detail methodologies for utilizing lanthanoids in characterizing protein complexes.
Main Methods:
- Protein sample preparation for lanthanoid binding.
- Execution of specialized NMR experiments to capture lanthanoid-induced signals.
- Interpretation of paramagnetic restraints (PCS, PRDC, PRE) for structural and dynamic information.
Main Results:
- Demonstration of lanthanoids as effective probes for protein structure elucidation via NMR.
- Correlation between lanthanoid ion position and protein nuclear environments.
- Generation of detailed structural and dynamic information through paramagnetic restraints.
Conclusions:
- Lanthanoids are powerful tools for characterizing lanthanoid-binding proteins using NMR.
- Paramagnetic restraints derived from lanthanoids provide critical data for protein structure and dynamics.
- This chapter serves as a guide to the preparation, experimentation, and interpretation involved in lanthanoid-based structural biology.
Related Concept Videos
01:14NMR Spectroscopy of Aromatic Compounds
5.5K
Aromatic compounds can be identified or analyzed using proton NMR and carbon‐13 NMR. Typically, aromatic hydrogens or hydrogens directly bonded to the aromatic rings are strongly deshielded by the aromatic ring current. Therefore, they absorb in the range of 6.5–8.0 ppm in proton NMR spectra. For instance, aromatic hydrogens directly bonded to the benzene ring absorb at 7.3 ppm. However, aromatic hydrogens of larger rings absorb farther upfield or downfield than the ideal range.
5.5K
01:25Applications Of NMR In Biology
4.1K
Nuclear magnetic resonance (NMR) spectroscopy is a very valuable analytical technique for researchers. It has been used for more than 50 years as an analytical tool. F. Bloch and E. Purcell formulated NMR in 1946 and won the 1952 Nobel Prize in Physics for their work. Biological macromolecules such as proteins, nucleic acids, lipids, and organic molecules including pharmaceutical compounds, can be studied using this versatile tool that exploits the magnetic properties of certain nuclei.
4.1K
01:02¹H NMR: Interpreting Distorted and Overlapping Signals
1.2K
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...
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...
1.2K
01:15¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR
1.3K
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.
1.3K
02:18The Equilibrium Binding Constant and Binding Strength
14.4K
The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
14.4K
01:08NMR Spectroscopy: Spin–Spin Coupling
2.5K
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...
2.5K