Summary of "Week 12 - Lecture 58"
Summary of Week 12 - Lecture 58: Basics and Techniques of Solid State NMR Spectroscopy in Structural Biology
Main Ideas and Concepts
Introduction to Solid State NMR (ssNMR) for Biological Samples
- ssNMR is ideal for studying biological samples that are difficult to crystallize or dissolve, such as integral membrane proteins and amyloid fibers.
- These samples are not amenable to X-ray crystallography or liquid state NMR.
Magic Angle Spinning (MAS)
- Samples are packed in rotors made of zirconium oxide and spun rapidly at the “magic angle” (~54.7°) relative to the magnetic field.
- MAS averages out anisotropic chemical shifts and dipolar couplings, dramatically improving spectral resolution and sensitivity.
- Different rotor sizes (7 mm, 4 mm, 3.2 mm, 2.5 mm, 1.3 mm, 0.8 mm) allow different spinning speeds, from ~5 kHz up to 120 kHz for proton detection.
Sample Preparation and Rotor Handling
- Biological samples are carefully packed into rotors with caps made of Kel-F or Vespel.
- Proper balancing and sealing are critical to prevent rotor breakage during high-speed spinning.
- Rotors spin using air-driven systems with drive and bearing pressures controlled in the probe assembly.
Decoupling
- Heteronuclear decoupling (e.g., proton decoupling during carbon detection) reduces dipolar couplings, improving resolution.
- Applying high-power decoupling pulses during detection narrows spectral lines.
Cross Polarization (CP)
- CP transfers polarization from abundant protons to less sensitive nuclei like carbon-13, enhancing sensitivity.
- Requires matching Hartmann-Hahn conditions (matching spin-lock fields of proton and carbon).
- CP is a fundamental pulse sequence in ssNMR.
Trade-off Between Resolution and Structural Information
- MAS and decoupling improve resolution but remove important structural interactions such as chemical shift anisotropy, dipolar couplings, and quadrupolar couplings.
- To retrieve these lost interactions, recoupling techniques are employed.
Recoupling Techniques
- Recoupling selectively reintroduces specific spin interactions that MAS and decoupling average out.
- Methods include:
- Rotary Resonance Recoupling: Matching spinning speed to chemical shift differences to reintroduce interactions.
- REDOR (Rotational Echo Double Resonance): Uses a series of 180° pulses synchronized with rotor periods to recouple heteronuclear dipolar interactions.
- Other sequences: PDSD (Proton Driven Spin Diffusion), DARR (Dipolar Assisted Rotational Resonance), RFDR (Radio Frequency Driven Recoupling).
Spin Diffusion and Polarization Transfer
- Proton-driven spin diffusion (PDSD) enables magnetization transfer between carbons via dipolar couplings.
- This facilitates homonuclear correlation experiments for structural information.
- Magnetization mixing and transfer can be controlled by mixing times and RF pulse schemes.
2D Correlation Experiments
- Extending 1D experiments into 2D enables correlation between different nuclei (e.g., C-C, N-C, H-N).
- Typical pulse sequence stages:
- Preparation (polarization transfer)
- Evolution (indirect dimension)
- Mixing (magnetization transfer)
- Detection (direct dimension)
- 2D spectra resemble liquid state HSQC or NOESY spectra but with broader lines due to residual dipolar couplings.
- Examples:
- PDSD or DARR: Carbon-carbon correlation spectra.
- NC Correlation: Nitrogen-carbon correlation similar to HSQC.
- H-N and C-H correlations: Show broader lines but useful for assignments.
Resonance Assignment and Structural Interpretation
- Sequential assignment strategies similar to liquid NMR exist for ssNMR (e.g., NCACB, NCOCA, etc.).
- Chemical shift deviations from random coil values provide secondary structure information (e.g., alpha helix vs beta sheet).
- ssNMR can thus be used for detailed structural and dynamic studies of insoluble or non-crystalline biological samples.
Future Directions
- Upcoming lectures will cover detailed pulse sequences and strategies for resonance assignment.
- Further discussion on extracting structural and dynamic parameters from ssNMR data.
Detailed Methodologies and Instructions
Magic Angle Spinning (MAS)
- Pack sample in zirconium oxide rotor.
- Cap rotor tightly with Kel-F or Vespel caps.
- Insert rotor into stator oriented at 54.7° to magnetic field.
- Spin rotor at appropriate speed (5 kHz to 120 kHz depending on rotor size).
- Use air pressure (drive and bearing) to maintain spinning.
Cross Polarization (CP) Sequence
- Apply 90° pulse to proton spins.
- Match Hartmann-Hahn condition (Ω_S γ_S = Ω_I γ_I) for polarization transfer.
- Cross-polarize magnetization from proton to carbon.
- Detect carbon signal while decoupling protons.
Decoupling
- During carbon detection, apply continuous high-power proton decoupling.
- This removes proton-carbon dipolar couplings, sharpening carbon peaks.
Recoupling Techniques
- Rotary Resonance: Adjust spinning speed to match chemical shift differences to reintroduce dipolar couplings.
- REDOR:
- Apply 90° proton pulse.
- Apply series of 180° pulses on carbon channel synchronized with rotor period.
- Refocus chemical shift Hamiltonian with mid-block 180° pulses.
- PDSD/DARR/RFDR:
- Use proton-driven spin diffusion or RF pulse trains to transfer magnetization between carbons.
- Control mixing time and RF power to tune correlation lengths.
2D Correlation Experiment Protocol
- Start with proton polarization.
- Transfer magnetization to carbon via CP.
- Allow evolution of spins during indirect dimension time (T₁).
- Apply mixing period (PDSD/DARR) for magnetization transfer.
- Detect carbon signal with proton decoupling.
- Repeat with incremented evolution times for 2D data.
- Perform 2D Fourier transform to obtain correlation spectra.
Resonance Assignment Strategy
- Use heteronuclear correlation spectra (NCACB, NCOCA, etc.).
- Assign peaks to amino acid residues based on known chemical shifts.
- Identify secondary structure elements by comparing chemical shifts to random coil values.
Speakers / Sources Featured
- Primary Speaker: The lecturer presenting the solid state NMR concepts and techniques (name not provided).
- No other speakers or external sources explicitly identified.
Summary Conclusion
This lecture provides a comprehensive overview of the fundamental principles and advanced techniques in solid state NMR spectroscopy as applied to structural biology. It covers sample preparation, magic angle spinning, cross polarization, decoupling, and recoupling methods essential for enhancing spectral resolution and sensitivity. The lecture also explains how these techniques enable 2D correlation experiments and resonance assignments, ultimately facilitating the determination of protein secondary structure and dynamics in non-crystalline biological samples.
Category
Educational
