Summary of "Week 4 - Lecture 20"

Summary of Week 4 - Lecture 20

This lecture focuses on advanced Nuclear Magnetic Resonance (NMR) spectroscopy techniques, particularly two-dimensional (2D) experiments that explore different mechanisms of magnetization transfer between nuclear spins. The main emphasis is on Nuclear Overhauser Effect Spectroscopy (NOESY) and its applications in structural biology, followed by an introduction to heteronuclear correlation experiments.

Main Ideas and Concepts

1. Recap of J-Coupling Based 2D NMR Experiments

Previous lectures covered 2D NMR experiments relying on J-coupling (scalar coupling) for magnetization transfer.
Examples include:
- COSY (Correlation Spectroscopy)
- Double Quantum Filtered COSY
- TOCSY (Total Correlation Spectroscopy)
- J-scaled and constant-time COSY variants
These experiments transfer information through scalar couplings between spins.

2. Introduction to Dipolar Interaction Based Transfer: NOESY

NOESY (Nuclear Overhauser Effect Spectroscopy) is based on dipolar interactions rather than J-coupling.
Also called exchange spectroscopy (EXSY or XC) due to the exchange mechanism of magnetization transfer.
NOESY is widely used for structural determination of molecules, especially large biomolecules.
EXSY is limited to systems with actual chemical exchange between conformations.

3. NOESY Pulse Sequence and Mechanism

The pulse sequence consists of three 90° pulses with a mixing time (τ_m) between the second and third pulses.
Magnetization evolution:
- Starts with transverse magnetization after the first 90° pulse.
- Frequency labeling during t₁ evolution.
- Second 90° pulse inverts z-magnetization.
- During mixing time, relaxation transfers magnetization between spatially close spins (dipolar coupling).
- Third 90° pulse converts z-magnetization back to transverse magnetization for detection.
Result: 2D spectrum with diagonal peaks (same spin) and cross peaks (between interacting spins).

4. Interpretation of NOESY Spectra

Cross peak intensity depends on:
- Mixing time (τ_m)
- Spin-lattice relaxation time (T₁)
- Spectrometer frequency (ω₀)
- Correlation time (τ_c) related to molecular motion
NOE sign depends on the product ω₀τ_c:
- Small ω₀τ_c (small molecules): Positive NOE
- Large ω₀τ_c (large molecules): Negative NOE
Cross peak signs and intensities provide insights into molecular size and dynamics.

5. Distance Dependence and Structural Information

NOE intensity ∝ 1 / r⁶ (r = inter-proton distance)
Strong NOE signals indicate short distances (< 5 Å), critical for determining 3D structures.
Steric constraints prevent distances below ~2.4 Å.
NOESY provides spatial proximity information useful for:
- Determining dihedral angles and conformations (e.g., α-helix vs β-sheet)
- Sequence-specific resonance assignments when combined with COSY

6. Sequential and Long-Range NOE Connectivities in Peptides/Proteins

NOESY can detect:
- Intra-residue correlations (within the same amino acid)
- Sequential correlations (between neighboring residues i and i±1)
- Long-range correlations (between residues far apart in sequence but close in space due to folding)
This allows mapping of the polypeptide chain and understanding protein folding.
NOESY spectrum is often called the “fingerprint” of the molecular structure.

7. Challenges and Complexity of Protein NOESY Spectra

Protein NOESY spectra contain thousands of peaks, including:
- Diagonal peaks (self-correlations)
- Cross peaks (short and long-range interactions)
- Overlapping signals due to crowded spectra
Quantitative analysis of peak intensities enables distance restraints for molecular modeling and structure calculation.

8. Introduction to Heteronuclear Correlation Experiments

Proton spectra can be crowded with strong diagonal peaks, making analysis difficult.
Heteronuclear correlation experiments (e.g., HSQC: Heteronuclear Single Quantum Coherence) correlate protons with other nuclei like ^13C or ^15N.
These experiments simplify spectra and provide clearer cross peak information.
Typically require isotopic labeling (^13C, ^15N) of proteins, which is now feasible due to advances in molecular biology (e.g., bacterial expression systems).
Heteronuclear experiments are crucial for studying larger biomolecules.

Methodology / Experimental Details (NOESY Pulse Sequence)

Pulse sequence steps:
1. 90° pulse → creates transverse magnetization.
2. Evolution during t₁ → frequency labeling.
3. Second 90° pulse → converts transverse magnetization to inverted z-magnetization.
4. Mixing time (τ_m) → magnetization transfer via dipolar interaction or chemical exchange.
5. Third 90° pulse → converts z-magnetization back to transverse magnetization.
6. Detection during t₂ → frequency labeling of transferred magnetization.
Data processing: 2D Fourier transform → diagonal and cross peaks.

Applications Highlighted

Structural determination of peptides and proteins.
Sequence-specific resonance assignments using combined COSY and NOESY data.
Identification of spatial proximity and folding patterns in polypeptides.
Differentiation of molecular size and dynamics via NOE sign and intensity.
Use of heteronuclear correlation experiments for simplifying spectra and studying larger biomolecules.

Speakers / Sources

Primary Speaker: Lecturer delivering Week 4 - Lecture 20 (name not provided).
No other speakers or external sources explicitly mentioned.

This lecture provides a foundational understanding of NOESY and its critical role in biomolecular NMR, setting the stage for further exploration of heteronuclear correlation techniques in subsequent classes.