NMR Lecture 6 Flashcards
What is the primary advantages of multinuclear, multidimensional NMR over 2D 1H NMR?
It allows unique identification of sites in the molecule using pairs of frequencies (1H/13C, 1H/15N, 13C/13C, 13C/15N) Different nuclei have distinct chemical shifts and offer additional information. This enhances the resolution and aids in the identification of specific functional groups within a molecule.
List two stable isotopes that are often needed to be enriched in the protein sample in a multinuclear NMR experiment. Why do they need to be enriched?
1) 13C: natural abundance in proteins is low. Enrichment of this is beneficial for improving the sensitivity of NMR experiments.
2) 15N: Natural abundance in proteins is low. Enrichment is crucial for studying protein structures and dynamics in biomolecular NMR.
Isotopic enrichment enhances the sensitivity of NMR experiments. It allows for the observation of weaker signals. Enrichment also increases the signal intensity of the nuclei of interest relative to the background noise, important for obtaining high-quality spectra.
List 4 parts that a 2D 1H-15N HSQC pulse sequence is composed of.
1) INEPT (Insensitive Nuclear Enhancement by Polarization Transfer): magnetization transfer from 1H to 15N
- selectively enhances the sensitivity of heteronuclear experiments
2) Magnetization of 15N is frequency labeled during t1
- allows the 15N nuclei to evolve in the magnetic field, encoding their chemical shift information
3) Reverse INEPT: magnetization transfer from 15N back to 1H
- essential for enhancing the sensitivity of 1H detection in the subsequent stages of the pulse sequence
4) Detection of FID by the receiver during t2
- Decoupling pulse is applied to 15N
- resulting 2D spectrum provides cross-peaks representing correlations between 1H and 15N chemical shifts.
What information does a 2D 1H-15N HSQC NMR spectrum provide?
1) Chemical shifts: the cross-peak positions are the chemical shifts of the 1H and 15N nuclei
- can provide information about the types of atoms and their surroundings in the molecule
2) Correlation Peaks: can reveal protons directly attached to specific nitrogen atoms in the backbone of a protein
3) Protein Dynamics: intensity and linewidth of the cross peaks provide insights into flexibility/dynamics
- broader = higher flexibility, sharper = rigid
8) Quality Control: NMR sample quality can be assessed by appearance and dispersion of cross-peaks
Why do you need to apply a decoupling pulse on 15N during t2?
It simplifies the observed spectrum and improves the quality of the data.
1) Resolution enhancement: 15N nuclei evolve in the presence of 1H-15N coupling interactions. Without decoupling, it can lead to complex splitting patterns and overlapping peaks in the 15N dimension, reducing spectral resolution.
2) 1H-15N coupling can introduce phase distortions: decoupling helps eliminate phase distortions, resulting in a more reliable spectrum.
What will the peak pattern become if there is no such decoupling applied?
1) Multiplet patterns: peaks in the 15N dimension would exhibit multiplet patterns, reflecting the coupling with neighboring 1H nuclei.
- # of peaks = # of equivalent protons coupled to a given 15N nucleus
2) Splitting due to coupling constants: size and pattern of splitting is influenced by the coupling constants between 1H and 15N nuclei
- larger coupling constants lead to larger splitting
3) Overlapping peaks: Coupling-induced splittings could lead to overlapping peaks, making it difficult to distinguish individual 15N resonances
- overlapping peaks hinder accurate peak assignment and compromise the ability to extract meaningful structural and dynamic information
4) Loss of resolution: coupling effects without decoupling lead to broader peaks and less spectral resolution.
- hard to determine chemical shifts and can obscure subtle features in the spectrum.
What information do the CBCA(CO)NH experiments provide?
only inter-residue connectivity
1) CO-N Connectivity: establishes connectivity between the backbone carbonyl carbon and the amide proton of the same amino acid residue. It correlates the carbonyl carbon of a residue with the amide proton of the same residue.
2) Sequential connectivity: provides sequential connectivity information, linking adjacent amino acid residues.
3) Chemical Shift information: provides information about chemical shifts of the carbonyl carbon and the amide proton, aiding in resonance assignment
What information do the 3D HNCACB experiments provide?
both intra and inter-residue connectivity
1) HN Connectivity: establishes connectivity between the amide proton and the alpha, beta, carbonyl carbons of the amino acid residues in a protein
2) Sequential Connectivity: crucial for determining amino acid sequence of the protein
3) Chemical shift information: each cross-peaks in the spectrum provides information about the chemical shifts of the correlated nuclei, aiding in the assignment of resonances
What information do both the 3D HNCACB and CBCA(CO)NH experiments provide?
1) Sequential Assignment: backbone resonances in a protein. We can trace the connectivity along the polypeptide chain.
2) Torsion Angle Constraints: The combination of these experiments helps derive torsion angle constraints, essential for structural calculations and determining the 3D structure of a protein
3) Structural Analysis: provides basis for building up the connectivity network in the protein structure
Describe how to achieve protein backbone chemical shift assignment using the combination of these two NMR spectra.
1) Data acquisition: get good 3D HNCACB and CBCA(CO)NH NMR spectra
2) Peak Picking: Identify peaks in both spectra, corresponding to specific NH or NCA bond in the backbone
3) Peak Matching: matcha corresponding peaks between both spectra based on their chemical shifts (usually represent the same amino acid residue in the sequence)
4) Sequential connectivity: utilize the sequential connectivity information to establish the amino acid sequence of the protein
5) Assignment Iteration: Use the connectivity information to assign chemical shifts to the adjacent residues iteratively
6) Resonance dispersal: chemical shifts of different types of amino acids exhibit characteristic dispersion patterns
Describe what NMR experiments are generally used for chemical shift assignment of protein side chains.
BACKBONE CHEMICAL SHIFT ASSIGNMENT: contributes to sequential backbone assignment by analyzing respective atoms
- 2D 1H-15N HSQC
- 3D HNCO
- 3D HNCACA
- 3D CBCA(CO)NH
SIDE CHAIN CHEMICAL SHIFT ASSIGNMENT: correlates aliphatic side-chain protons with their directly bonded carbon atoms
- 2D 1H-13C HSQC
- 3D HBHA(CO)NH
- 3D H(CCO)NH
- 3D C(CO)NH
- 3D HCCH-TOCSY
NOE ASSIGNMENT: utilizes NOE to provide distance constrains for atoms with their respective atom
-3D 15N-edited [1H, 1H] NOESY-HSQC
- 3D 13C-edited [1H, 1H] NOESY-HSQC (aliphatic)
- 3D 13C-edited [1H, 1H] NOESY-HSQC (aromatic)
