NMR Lecture 7

Question 1

What information on a protein system can be inferred by chemical shifts?

Answer 1

1) Secondary structure: chemical shifts of backbone nuclei are sensitive to local structural elements
2) Residue-specific information: chemical shifts are specific to individual amino acid residues, allowing for the identification and assignment of resonances to specific positions in the protein sequence
3) Hydrogen bonding: chemical shifts of certain nuclei are influenced by hydrogen bonding interactions

Question 2

Describe how to use chemical shifts perturbation method to map protein interaction site.

Answer 2

1) isotope labeling
2) record high-resolution NMR spectra of the labeled protein to establish a baseline
3) add the interacting partner to the protein solution
4) record NMR spectra of the proton in the presence of the interacting partner
5) Compare chemical shifts of specific nuclei between free and bound states
6) Calculate chemical shift perturbation values for each nucleus to quantify changes
7) create a plot of CSP values against residue numbers to identify perturbed regions.

Question 3

How does a 2D 1H, 15N-HSQC experiment provide protein folding and protein interaction information?

Answer 3

1) Chemical shifts: sensitive to the local electronic environment, providing information about the secondary structure elements and overall protein folding
2) Chemical Shift Perturbations: Significant deviations in peak positions can be analyzed to identify residues involved in the interaction
3) Dissociation constants: provides quantitative information about the strength of the interaction

Question 4

In comparison with 1D 1H NMR, what advantages does 2D 1H, 15N-HSQC offer?

Answer 4

1) Resonance assignment: each peak corresponds to a unique pair of 1H and 15N nuclei in the protein backbone
- 1D 1H NMR may have overlapping peaks
2) Structural information: 2D provides heteronuclear correlation between 1H and 15N nuclei, yielding structural information about the protein, like secondary structure elements
3) CSP: enables mapping of binding interfaces and provides insights into the molecular details of the interaction
- 1D spectrum isn’t as detailed about binding sites
4) Dissociation Constants: by gradually adding an interacting partner allows for the determination of Kd

Question 5

What is secondary chemical shift?

Answer 5

it’s the difference between observed chemical shifts and random coil chemical shifts.
It helps identify specific patterns associated with different structural elements in proteins.

Question 6

What nuclei’s chemical shifts were taken as input information for CSI? What structural information does CSI provide?

Answer 6

CSI (Chemical Shift Index): used in protein NMR spectroscopy and relies on chemical shifts of backbone nuclei. Uses HN and N15 in the protein backbone, commonly found in 2D 1H-15N HSQC
- Alpha-helix: positive CSI values for amide protons and nitrogen atoms
- Beta-sheet: negative CSI values for amide protons and nitrogen atoms
- random coil: CSI values close to zero

Question 7

What nuclei’s chemical shifts were taken as input information for TALOS? What structural information does TALOS provide?

Answer 7

TALOS (Torsion Angle Likelihood Obtained from Shifts): used in protein NMR spectroscopy to predict protein backbone torsion angles based on chemical shift information. Uses HN, N15, CA, CB, C’ observed in 2D and 3D NMR
- Phi (ϕ): torsion angle around CA-CN bond
- Psi (ψ): torsion angle about CN-CA bond
- Theta (θ): angle between CA-CN bond vector and NH bond vector
- Omega (ω): torsion angle around the N-CA bond

Question 8

Discuss the pros and cons of NMR spectroscopy in macromolecular structure determination.

Answer 8

Pros:
- NMR provides structural information in solution, allowing for dynamic processes and interactions under near-physiological conditions
- NMR can provide details about protein dynamics, flexibility, and changes over time
- NMR doesn’t require crystallization
- NMR is applied to small proteins (up to 50 kDa) and nucleic acids
- NMR is sensitive to hydrogen bonds, allowing for direct observation in protein structure

Cons:
- harder to analyzing larger macromolecules due to signal broadening
- harder to achieve high-resolution structures, especially for larger proteins
- lower sensitivity than x-ray crystallography
-Signals overlap in NMR and can complicate resonance assignment

Question 9

Discuss the pros and cons of x-ray crystallography in macromolecular structure determination.

Answer 9

Pros:
- provides high-resolution structures and gives detailed insights in the geometry
- can handle larger molecules and can use in a wide range of biological system
- well-established and widely adopted technique

Cons:
- requires growth of high-quality crystals (can be hard for some proteins)
- static snapshot of the structure, no dynamic movement
- can introduce artifacts, leading to non-physiological structures
- x-ray radiation can can damage to the crystal