NMR Lecture 8

Question 1

List and describe the most commonly used experimental restraints for NMR structure calculation.

Answer 1

1) NOE: obtained from 2D and 3D 1H, 1H-NOESY experiments.
- provide distance information between pairs of atoms; crucial for defining the spatial arrangement of atoms in the molecule
2) Dihedral angles: derived from experimentally measured spin-spin couplings or predicted from TALOS
- provide information about the backbone and side-chain torsional angles, aiding in the determination of the protein’s overall conformation
3) Hydrogen Bond: derived from the experimentally observed 3hJ(NC) couplings or inferred from NOE restraints or secondary structure information
- help define the presence and geometry of hydrogen bons in the protein structure, providing insights into secondary structure elements
4) Residual Dipolar Couplings: measured from a difference in the coupling constants between aligned and unaligned media
- provide orientational information, helping to determine the relative orientation of different structural elements in the molecule

Question 2

Describe the general procedure for structure determination of a 20 kD monomeric protein by NMR spectroscopy.

Answer 2

1) Protein expression and purification: purify protein in high homogeneity using chromatographic techniques
2) Protein isotopic labeling: incorporate isotopic labels into the protein to enhance NMR sensitivity
3) NMR Sample preparation: dissolve isotopically labeled protein in NMR buffer
4) Data Collection: get multidimensional NMR spectra
5) Resonance: assign NMR signals to specific atoms in the protein Assignment
6) Distance Restraint generation: classify NOES into short, medium, long range based on the observed signal intensities
7) Torsion Angle Restraint Calculation: derive torsion angle restraints from J-coupling data
8) Structural calculation: use specialized software packages
9) Structure refinement: refine calculated structures using molecular dynamics simulations or energy minimization protocols

Question 3

What information is taken as input for automated NOE assignment?

Answer 3

• Amino acid sequence: provides primary structure of the protein
• Sequence-specific assignments: connect the observed NMR signals to the corresponding amino acids in the protein
• NOESY cross peak positions and volumes: positions of these peaks provide information about the interacting protons, while the volumes of the peaks indicate the strength of the interactions

Question 4

What are the input requirements for a successful automated NOE assignment?

Answer 4

Completeness of chemical shift assignments:
- provide reference for the positions of peaks in the NOESY spectrum, facilitating the assignment of NOE
- incomplete chemical shift assignments can lead to ambiguities in peak identification
Quality of NOE peaks in long range ones:
- strength and accuracy influence the reliability of distance information used in structure calculations

Question 5

What is the output information from the automated NOE assignment?

Answer 5

• NOE Assignments: provides a detailed mapping of NOE within the protein, establishing spatial relationships between specific protons
• 3D structures: helps generate a 3D model of the biomolecule

Question 6

Briefly explain what is simulated annealing. What kind of structure calculation issue does a simulated annealing protocol help to overcome?

Answer 6

Simulated annealing: starting structure is heated to a high temperature in a simulation, followed by slow cooling steps
- during many discrete cooling steps the starting structure can evolve towards the energetically favorable final structure under the influence of a force field derived from the constraints

Question 7

List the five parameters that are commonly used for evaluating the quality of NMR structures.

Answer 7

1) molecular energies: help assess the stability and energetics of a structure
2) number of experimental constraints: reflects the amount of information used in structure calculations
3) root-mean-square deviation of structural ensemble: represents the average deviation between different structures within the ensemble
4) violation of constraints/restraints: fewer violations indicate better agreement between the calculated structure and the experimental data
5) comparison to known structures: PROCHECK: can provide additional validation. PROCHECK assesses the geometry of protein structures