Summary of "Week 5 - Lecture 23"

Summary of “Week 5 - Lecture 23”

This lecture focuses on advanced multidimensional NMR experiments, particularly triple resonance techniques developed primarily for studying protein structures. The main emphasis is on how magnetization transfer pathways in these experiments enable sequential resonance assignments crucial for structural biology.

Main Ideas and Concepts

Motivation and Application of Triple Resonance NMR

Triple resonance NMR experiments were developed largely due to the need for detailed structural information on proteins.
These methods have been instrumental in structural biology, especially for proteins, though they also apply to nucleic acids and other biomolecules.
The development of new pulse sequences is driven by the desire to improve protein structure determination.

Magnetization Transfer Pathways

Magnetization transfer occurs through the polypeptide backbone and sometimes side chains.
Transfers are based on J-couplings (mostly one-bond couplings, some two-bond), which are fairly consistent in magnitude (~4–9 Hz).
The constant-time HNCA experiment is explained as an example:
- Magnetization flows from the amide proton (HN) → nitrogen (^15N) → alpha carbon (Cα) → back to nitrogen → back to proton for detection.
- Chemical shift labeling occurs during incremental time periods (t₁, t₂, t₃) corresponding to ^15N, Cα, and HN respectively.
- After 3D Fourier transformation, spectra show correlations between these nuclei.

Sequential Assignment Using HNCA

The HNCA spectrum contains two peaks per amide proton chemical shift:
- A self peak from the same residue’s Cα.
- A sequential peak from the previous residue’s (i-1) Cα.
By scanning through the ^15N dimension, one can “walk” along the polypeptide chain by connecting sequential peaks.
Differences in coupling constants and peak intensities help distinguish self and sequential peaks.

HNN Experiment

Magnetization transfer pathway: amide proton → nitrogen → Cα of residues i and i-1 → back to nitrogen of i, i-1, and i+1 → amide protons.
The 3D spectrum has two nitrogen dimensions (t₁ and t₂) and one proton dimension (t₃).
At a given nitrogen chemical shift, three peaks appear corresponding to residues i, i-1, and i+1 (triplet filter).
This allows simultaneous identification of neighboring residues, facilitating sequential assignment.
The sign (phase) of peaks varies depending on residue type and sequence context, providing checkpoints during assignment.
Presence of glycine or proline residues alters the peak patterns and intensities, aiding in sequence identification.

HN(C)N Experiment

Magnetization pathway: amide proton → nitrogen → carbonyl of i-1 → Cα of i-1 → nitrogen of i-1 and i → amide proton.
Produces a 3D spectrum with two nitrogen dimensions and one proton dimension.
Provides a “doublet filter” showing correlations between residues i and i-1 or i and i+1 depending on the plane.
Peak sign patterns differ from HNN, allowing determination of directionality along the polypeptide chain.
This directionality is crucial for unambiguous sequential resonance assignments.

Significance of Peak Patterns and Signatures

Positive and negative peak signs correlate with residue type and sequence order.
Glycine residues cause distinctive sign changes, serving as checkpoints.
Proline residues lack amide protons, leading to missing peaks and helping identify sequence breaks.
These patterns improve confidence in assignments and help detect errors.

Applications and Advantages

These triple resonance experiments enable rapid and unambiguous resonance assignments, even in intrinsically disordered proteins or proteins with flexible regions.
Nitrogen chemical shift dispersion is generally better than carbon alpha, aiding in spectral resolution.
These methods are valuable in studying protein folding, unfolding, and aggregation pathways.
The lecture emphasizes methodological development driven by practical challenges in protein NMR.

Methodologies / Instructions

1. Constant-Time HNCA Experiment

Start magnetization on amide proton (HN).
Transfer magnetization to nitrogen (^15N) via INEPT.
Hold magnetization on nitrogen during t₁ increment (labels ^15N chemical shifts).
Transfer magnetization to Cα (carbon-13 alpha).
Use t₂ increment to label Cα chemical shifts.
Transfer magnetization back to nitrogen and then to proton for detection.
Detect proton chemical shifts during t₃.
Perform 3D Fourier transform to obtain a spectrum with axes: F₁ = ^15N, F₂ = Cα, F₃ = HN.
Identify self and sequential peaks by analyzing peak positions and intensities.

2. HNN Experiment

Magnetization transfer: HN → ^15N → Cα (residues i and i-1) → ^15N (i-1, i, i+1) → HN (i-1, i, i+1).
Use two nitrogen dimensions (t₁ and t₂) and one proton dimension (t₃).
After 3D Fourier transform, analyze planes at fixed ^15N chemical shifts.
Identify triplets of peaks corresponding to three sequential residues.
Use peak sign patterns to detect glycine residues and confirm sequential assignments.
Note missing peaks if proline is present (no amide proton).

3. HN(C)N Experiment

Magnetization transfer: HN → ^15N → carbonyl (i-1) → Cα (i-1) → ^15N (i-1 and i) → HN.
Two nitrogen dimensions and one proton dimension.
After 3D Fourier transform, analyze cross-sections:
- F₁–F₃ plane shows peaks for residues i and i+1.
- F₂–F₃ plane shows peaks for residues i and i-1.
Use doublet filter pattern to identify sequential residues.
Peak sign patterns provide directionality along the chain.
Use these patterns to confirm assignments and detect errors.

4. Sequential Walk Strategy

Use HNCA to identify Cα chemical shifts and walk through residues by connecting sequential peaks.
Use HNN to obtain triplet correlations and confirm neighboring residues.
Use HN(C)N to determine directionality and resolve ambiguities.
Use peak sign patterns, presence of glycines and prolines as checkpoints.
Combine data from multiple experiments for robust resonance assignment.

Speakers / Sources

The lecture is delivered by a single unnamed instructor (likely a professor or researcher in NMR spectroscopy).
Proteins such as FKBP and ubiquitin are referenced as examples.
No other speakers are featured.

Overall, this lecture provides an in-depth explanation of triple resonance NMR experiments (HNCA, HNN, HN(C)N) used for sequential resonance assignment in proteins, emphasizing magnetization transfer pathways, spectral interpretation, and the use of peak patterns and signs to facilitate rapid, accurate protein structure determination.