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                     Bio 5476 Protocol for Lab Exercise #4
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 (1) Download the PDB structure of the trypsin-benzamidine complex (1CE5),
     and  observe the binding site for the benzamidine ligand. Use the
     TINKER "pdbxyz" program to convert the trypsin structure to
     xyz format (the benzamidine will be lost in this process).

 (2) Obtain the TINKER xyz file for benzamidine from the "MOLECULES" area
     on the lab web site. That directory also contains a modified version
     of the OPLS-AA parameters (ie, oplsaa.prm) with  missing parameter
     values added for benzamidine.

 (3) Perform conformational analysis of benzamidine to find the barrier
     to rotation for the bond connecting the ring to the amidine. What
     is the preferred conformation? Does your analysis agree with the
     structure contained in the PDB file. You can use the TINKER keyword
     RESTRAIN-TORSION to restrict the torsional angle to a desired value.
     For example, "restrain-torsion  1 2 3 4  10.0  120.0" will restrain
     the torsion defined by atoms 1-2-3-4 to an angle of 120 degrees
     with a restraining force constant of 10 kcal/mol/degree^2, which is
     quite a strong restraint.

 (4) Dock the benzamidine molecule into the trypsin structure. Use can
     use the TINKER "xyzedit" program from the command line to merge
     the trypsin xyz file with your benzamidine, once you get the ligand
     moved to the correct coordinates. The X-ray determined coordinates
     for the benzamidine non-hydrogen atoms, as taken from the 1CE5
     PDB file can be found in TINKER xyz file "benzamidine.xray" in
     the lab "MOLECULES" area.

 (5) Minimize the benzamidine while holding the protein rigid. What is
     the interaction energy between the protein and the ligand? Does the
     minimum energy structure you compute agree with the PDB structure?
     You can use the "ACTIVE" keyword to allow only the benzamidine
     atoms to move during minimization.

 (6) Perform an MD simulation, allowing only the benzamidine and a sphere
     of atoms within the protein binding site to move. A 15 Ang sphere
     centered on the amidine carbon is a reasonable choice. Analyze your
     trajectory to find the average conformation of the benzamidine
     molecule. Does this give better or worse agreement with the crystal
     structure?

 (7) This is a classic, much studied, example of a protein-inhibitor
     binding complex. Compare your results with those from the papers
     published by the Brooks group (PROTEINS, 58, 407-417, 2005) and
     by the Jorgensen group (J. Phys. Chem. B, 101, 9663-9669, 1997).