9.3 NMR spectroscopy in structural biology

NMR spectroscopy is a powerful tool in structural biology, using magnetic properties of atomic nuclei to reveal molecular structures. It provides detailed info on protein folding, dynamics, and interactions in solution, offering insights into biological functions.

Unlike X-ray crystallography, NMR studies proteins in their native environment. It excels at capturing weak interactions and protein flexibility. However, it's limited by protein size and sample requirements, making it best for smaller molecules.

Principles of NMR Spectroscopy

Fundamental Concepts

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• NMR spectroscopy is a non-destructive technique that exploits the magnetic properties of certain atomic nuclei (1H, 13C, 15N) to determine the physical and chemical properties of atoms or molecules
• The fundamental principle of NMR is based on the interaction between the magnetic moments of specific nuclei and an external magnetic field
• When placed in a strong magnetic field, NMR-active nuclei absorb and re-emit electromagnetic radiation at a specific resonance frequency, which depends on the strength of the magnetic field and the magnetic properties of the isotope

Chemical Environment and Structural Information

• The chemical environment of the nuclei influences the resonance frequency, leading to chemical shifts in the NMR spectrum that provide information about the local electronic and molecular structure
• In structural biology, NMR spectroscopy is used to determine the three-dimensional structures of proteins, nucleic acids, and their complexes in solution
• NMR can provide detailed information on the conformational dynamics, ligand binding, and intermolecular interactions of biomolecules, which is crucial for understanding their biological functions

Information from NMR Experiments

1D and 2D NMR Spectra

• One-dimensional (1D) NMR spectra provide information about the chemical shifts, coupling constants, and relative intensities of the NMR signals, which can be used to identify functional groups and local chemical environments
• Two-dimensional (2D) NMR experiments, such as COSY, TOCSY, and NOESY, correlate the chemical shifts of nuclei that are coupled through bonds (COSY, TOCSY) or space (NOESY), providing information about the connectivity and spatial proximity of atoms in a molecule
  • COSY (Correlation Spectroscopy) identifies coupled nuclei connected by up to three chemical bonds
  • TOCSY (Total Correlation Spectroscopy) identifies all nuclei within a spin system, regardless of the number of intervening chemical bonds
  • NOESY (Nuclear Overhauser Effect Spectroscopy) identifies nuclei that are spatially close (typically < 5 Å), even if they are not directly bonded

3D and Heteronuclear NMR Experiments

• Three-dimensional (3D) NMR experiments, such as HNCA, HNCO, and HCCH-TOCSY, are used to simplify the assignment of resonances in larger proteins by spreading the information across three frequency dimensions
  • 3D experiments often combine 2D NMR techniques with an additional frequency dimension to resolve overlapping signals and facilitate resonance assignments
• Heteronuclear NMR experiments, such as 1H-15N HSQC and 1H-13C HSQC, provide information about the correlation between protons and heteroatoms (15N or 13C) in isotopically labeled proteins, aiding in resonance assignments and structure determination
  • 1H-15N HSQC (Heteronuclear Single Quantum Coherence) experiments correlate the chemical shifts of directly bonded 1H and 15N nuclei, providing a fingerprint of the protein backbone
  • 1H-13C HSQC experiments correlate the chemical shifts of directly bonded 1H and 13C nuclei, providing information about the side-chain structure and connectivity

Interpreting NMR Spectra

Protein Folding and Secondary Structure

• The chemical shift dispersion in 1H-15N HSQC spectra can provide information about the overall folding state of a protein, with well-dispersed peaks indicating a folded structure and poorly dispersed peaks suggesting an unfolded or disordered state
• Secondary structure elements, such as α-helices and β-sheets, can be identified by characteristic patterns of NOE connectivities and chemical shifts in the NMR spectra
  • α-helices exhibit strong sequential HN-HN NOEs and medium-range HN-Hα NOEs (i to i+3, i+4)
  • β-sheets display strong sequential HN-Hα NOEs and long-range HN-HN NOEs between strands

Resonance Assignments and Tertiary Structure

• The sequential assignment of backbone resonances using a combination of 2D and 3D NMR experiments allows for the determination of the protein's primary structure and provides a foundation for structural analysis
  • Sequential assignments involve identifying the connectivity between neighboring amino acid residues based on characteristic patterns of NOE connectivities and chemical shifts
• Long-range NOE connectivities between protons that are close in space but far apart in the primary sequence are used to define the tertiary structure of a protein
  • These long-range NOEs provide distance restraints that can be used in structure calculation algorithms to generate a three-dimensional model of the protein

Dynamics and Interactions

• Conformational dynamics of proteins can be studied using NMR relaxation measurements, such as T1, T2, and heteronuclear NOE experiments, which provide information about the motions of individual atoms on various timescales
  • T1 (longitudinal) relaxation reflects the rate of recovery of magnetization along the external magnetic field and is sensitive to fast motions (ps-ns timescale)
  • T2 (transverse) relaxation reflects the rate of decay of magnetization perpendicular to the external magnetic field and is sensitive to slower motions (ns-ms timescale)
• NMR chemical shift perturbation experiments can identify binding interfaces and measure the affinity of protein-ligand or protein-protein interactions by monitoring changes in the chemical shifts of specific residues upon titration with a binding partner
  • Residues involved in the binding interface typically exhibit significant changes in chemical shifts upon complex formation
  • The magnitude of the chemical shift perturbations can be used to map the binding site and estimate the dissociation constant (Kd) of the interaction

NMR Spectroscopy: Advantages vs Limitations

Advantages in Structural Biology

• NMR allows for the study of proteins in solution, providing a more physiologically relevant environment compared to X-ray crystallography
  • Solution-state NMR enables the investigation of proteins under near-native conditions, including pH, temperature, and ionic strength
• NMR can provide detailed information on the conformational dynamics and flexibility of proteins, which is crucial for understanding their biological functions
  • NMR relaxation measurements and paramagnetic relaxation enhancement (PRE) experiments can probe protein dynamics on various timescales
• NMR is well-suited for studying weak or transient interactions, such as protein-ligand or protein-protein interactions, which may be difficult to capture using other techniques
  • NMR titration experiments can detect weak interactions with dissociation constants (Kd) in the micromolar to millimolar range
• Isotopic labeling strategies enable the study of specific regions or amino acid types within a protein, facilitating the investigation of complex systems
  • Selective labeling techniques, such as amino acid-specific labeling or segmental labeling, can simplify NMR spectra and focus on regions of interest

Limitations in Structural Biology

• NMR spectroscopy is generally limited to proteins smaller than 50-60 kDa due to signal overlap and decreased sensitivity for larger molecules. However, recent advances in NMR methodology and instrumentation have pushed this limit to larger systems
  • Deuteration, selective labeling, and higher magnetic field strengths have enabled the study of larger proteins and complexes
• NMR requires relatively large amounts of pure, isotopically labeled protein samples (typically 0.1-1 mM), which can be challenging to obtain for some proteins
  • Protein expression and purification can be time-consuming and costly, especially for proteins that are difficult to express or purify in large quantities
• Data acquisition and analysis can be time-consuming, especially for larger proteins or complex systems
  • NMR experiments for structure determination and dynamics studies often require multiple samples and long measurement times
• The interpretation of NMR data often relies on complex computational methods, such as molecular dynamics simulations and structure calculation algorithms, which can be computationally intensive and require specialized expertise
  • NMR structure determination involves the integration of various experimental restraints (NOEs, dihedral angles, hydrogen bonds) into molecular dynamics simulations or energy minimization protocols to generate an ensemble of structures consistent with the experimental data