[公告] NMR第二題-附完整解答!!!
2. Describe the key steps in using NMR to solve the structure of an FHA
domain, with ca. 120 residues, which can be over expressed in E. coli. There
is no standard answer to this question and the grading will be based on the
depth of your understanding. You should start with sample preparation and
include the final refinement.
Answer in English only. (20 points)
Sample preparation:
Since the FHA domain has 120 residues, we must label the protein while the
proteins are being synthesized in E.coli. We can label the protein with 15N
by replacing the ammonium chloride in the M9 medium with 15N labeled ammonium
chloride. Similarly, we can label the protein with 13C by replacing the
glucose in the M9 medium with 13C -labeled glucose. The protein can also be
doubly labeled with both 15N and 13C.
Structure determination:
1. Generation of molecular template by giving the computer the protein’s
primary sequence.
2. Collection of the 2D and 3D NMR spectrum.
3. Sequence specific total resonance assignment:
a. This includes:
- Back bone assignment
- Side chain assignment
b. The main aim is to find out the chemical shifts of all the
atoms in the protein.
c. Secondary structures of the protein can be extracted from this
information.
4. Determination of conformation constraints:
a. Finding the distance constraints between different atoms by
2D-NOESY:
- Only those two atoms that are close together in space can
contribute an NOE peak.
- The closer the distance between the two atoms, the
stronger the signal.
- NOE is inversely proportional to the distance. (NOE~1/r6)
- Usually NOE signals can be detected if the distance is
within 6 angstroms.
b. Finding the torsion angle constraints by measuring the
coupling constant:
- Based on the backbone chemical shift, dihedral angles
(phi and psi angles) can be derived.
c. Finding other constraints:
- Parameters like hydrogen bondings can be given. Hydrogen bonded
atoms are close together.
5. Initial Calculations:
a. Calculate the initial structure which has the lowest free energy.
b. Calculate distance geometry
c. Simulated annealing
Refinement:
a. The structural models obtained must overlap in 3 dimensions.
If not, more parameters must be given or that the parameters
must be modified.
b. Mixing time can also be changed in order to set the distance for
NOE signals to appear.
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