12.2 Nuclear magnetic resonance (NMR) spectroscopy fundamentals

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying biological molecules. It uses magnetic properties of atomic nuclei to reveal structures and interactions of proteins, nucleic acids, and metabolites at the atomic level.

NMR provides detailed info on chemical environments, connectivity, and spatial relationships of atoms in molecules. It's widely used to study protein structures, RNA conformations, and metabolic profiles in various biological contexts.

NMR Spectroscopy Basics

Principles and Applications

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• NMR spectroscopy is a non-destructive analytical technique that utilizes the magnetic properties of atomic nuclei to determine the structure, dynamics, and interactions of molecules
• The fundamental principle of NMR is based on the fact that certain atomic nuclei possess spin angular momentum and magnetic moments, which can interact with an external magnetic field
• When placed in a strong magnetic field, NMR-active nuclei (such as 1H, 13C, 15N, and 31P) can absorb and emit electromagnetic radiation at specific frequencies, known as the Larmor frequency
• The Larmor frequency depends on the strength of the external magnetic field and the gyromagnetic ratio of the nucleus, which is unique for each isotope

Biological Applications

• NMR spectroscopy can provide detailed information about the chemical environment, connectivity, and spatial proximity of atoms within a molecule
• In the context of biological molecules, NMR spectroscopy is widely used to study the structure, dynamics, and interactions of proteins, nucleic acids, and metabolites
  • Protein NMR can reveal the secondary and tertiary structure, conformational dynamics, and ligand-binding interactions of proteins in solution (e.g., determining the 3D structure of enzymes or identifying drug-binding sites)
  • NMR studies of nucleic acids can provide insights into their secondary structure, base-pairing interactions, and dynamics (e.g., investigating the conformational changes of RNA aptamers upon ligand binding)
  • Metabolomics NMR can identify and quantify various metabolites in biological samples, such as biofluids or tissue extracts (e.g., analyzing metabolic profiles in disease states or monitoring drug metabolism)

Chemical Shift in NMR

Definition and Origin

• Chemical shift is a fundamental concept in NMR spectroscopy that describes the variation in the resonance frequency of a nucleus due to its local chemical environment
• The chemical shift arises from the shielding or deshielding effects of the surrounding electrons on the nucleus, which alters its effective magnetic field
  • Shielding occurs when the local electron density around a nucleus opposes the external magnetic field, reducing the effective field experienced by the nucleus and resulting in a lower resonance frequency (upfield shift)
  • Deshielding occurs when the local electron density around a nucleus enhances the external magnetic field, increasing the effective field experienced by the nucleus and resulting in a higher resonance frequency (downfield shift)

Factors Influencing Chemical Shift

• The chemical shift is expressed in parts per million (ppm) relative to a reference compound, such as tetramethylsilane (TMS) for 1H and 13C NMR
• The chemical shift is highly sensitive to the local chemical environment, including factors such as the type of bonding, hybridization, electronegativity of neighboring atoms, and the presence of aromatic rings or hydrogen bonding
• In proteins, the chemical shifts of backbone and side-chain nuclei are influenced by the secondary structure (α-helices, β-sheets, and random coils) and the tertiary structure of the protein (e.g., the chemical shift of a glycine residue in an α-helix differs from that in a β-sheet)
• In nucleic acids, the chemical shifts of base and sugar protons are affected by the type of base (purine or pyrimidine), the glycosidic bond conformation (anti or syn), and the secondary structure (duplex, hairpin, or higher-order structures)

Spin-Spin Coupling in NMR

Definition and Origin

• Spin-spin coupling, also known as J-coupling or scalar coupling, is an interaction between the magnetic moments of two NMR-active nuclei mediated through chemical bonds
• Spin-spin coupling arises from the polarization of bonding electrons by the magnetic moments of the coupled nuclei, which results in a splitting of the NMR signal into multiple peaks
• The magnitude of the coupling, expressed as the coupling constant (J), depends on the number and type of intervening bonds, as well as the dihedral angle between the coupled nuclei

Multiplicity and Intensity

• The number of peaks in a multiplet is determined by the n+1 rule, where n is the number of equivalent coupled nuclei. For example, a proton coupled to two equivalent protons will appear as a triplet
• The intensity ratios of the peaks in a multiplet follow Pascal's triangle, such as 1:1 for a doublet, 1:2:1 for a triplet, and 1:3:3:1 for a quartet
• Spin-spin coupling provides valuable information about the connectivity and stereochemistry of molecules, as the coupling constants are sensitive to the dihedral angles between the coupled nuclei (Karplus relationship)
  • In proteins, spin-spin coupling between backbone amide protons and alpha protons (3JHN-Hα) can provide information about the secondary structure, as the coupling constants differ for α-helices (3-5 Hz), β-sheets (8-10 Hz), and random coils (6-8 Hz)
  • In nucleic acids, spin-spin coupling between sugar protons (3JH1'-H2') can indicate the sugar pucker conformation (C2'-endo or C3'-endo), which is related to the overall conformation of the nucleic acid (A-form or B-form)

Interpreting NMR Spectra

One-Dimensional NMR Spectra

• One-dimensional (1D) NMR spectra, such as 1H or 13C NMR, provide a simple representation of the chemical shifts and spin-spin coupling patterns of the nuclei in a molecule
  • In 1D protein NMR, the amide proton region (6-10 ppm) contains signals from the backbone amide protons, while the aliphatic region (0-5 ppm) contains signals from the side-chain protons
  • In 1D nucleic acid NMR, the base proton region (7-9 ppm) contains signals from the aromatic protons of the bases, while the sugar proton region (3-6 ppm) contains signals from the sugar protons

Two-Dimensional NMR Spectra

• Two-dimensional (2D) NMR experiments provide additional information by correlating the chemical shifts of two different nuclei, allowing for the assignment of resonances and the determination of structural constraints
  • Homonuclear 2D experiments, such as COSY (Correlation Spectroscopy) and TOCSY (Total Correlation Spectroscopy), correlate the chemical shifts of protons that are coupled through bonds, providing information about the connectivity of the molecule (e.g., identifying spin systems in proteins)
  • Heteronuclear 2D experiments, such as HSQC (Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Correlation), correlate the chemical shifts of protons with those of heteronuclei (e.g., 13C or 15N), providing information about the connectivity and the type of nuclei involved (e.g., identifying the amino acid type based on the 13C chemical shifts)
• In protein NMR, 2D experiments such as 1H-15N HSQC are used to obtain a "fingerprint" of the protein, where each peak represents a backbone amide group. Changes in peak positions or intensities can indicate changes in the structure or dynamics of the protein upon ligand binding or under different conditions (e.g., pH, temperature, or presence of denaturants)
• In nucleic acid NMR, 2D experiments such as NOESY (Nuclear Overhauser Effect Spectroscopy) are used to identify through-space interactions between protons, providing distance constraints for structure determination. Base-pairing interactions can be identified by the presence of cross-peaks between the imino protons of the bases (e.g., G-C base pairs)

Resonance Assignment and Structure Determination

• The interpretation of NMR spectra requires the assignment of resonances to specific nuclei in the molecule, which is achieved through a combination of 2D experiments and knowledge of the primary sequence of the protein or nucleic acid
• Advanced NMR techniques, such as triple-resonance experiments (e.g., HNCA, HNCACB) for protein backbone assignment and isotope-edited experiments (e.g., 13C-edited NOESY) for structure determination, further enhance the power of NMR in studying biological molecules
  • Triple-resonance experiments correlate the chemical shifts of the backbone amide proton, nitrogen, and carbon nuclei, allowing for the sequential assignment of the protein backbone
  • Isotope-edited experiments selectively detect NOE cross-peaks between protons attached to isotopically labeled nuclei (e.g., 13C or 15N), reducing spectral overlap and facilitating the assignment of side-chain resonances and the collection of distance constraints for structure calculation