BaMBEd

1. Molecular Mechanics and Dynamics Characterization of an In SilicoMutated Protein: A Stand-Alone Lab Module or Support Activity for In Vivo and In VitroAnalyses of Targeted Proteins BaMBEd

2. This week • Learn about the software for modeling • Use the software to create representations of your protein highlighting features of interest • Make a mutant form of your protein • Solvate your mutant protein • Start your mutant protein “shaking” to find configuration that gives you the energy minimum • Measure the ATP-binding pocket in wild type CK1

3. Your lab report • In your growing lab report, you’ll • Make revisions • Report the modeling you set up this week (not in detail) • Report the size of the ATP-binding pocket in the wild type CK1 (probably in table form) • If you don’t get these measurements finished this week, you can come in and use the computers next week to finish. Lab report due in two weeks.

4. Downloading the CK1 Structure • Set up a folder to do your work in • C:/Users/Student/My Documents/CK1-modeling • We are going to use a crystal structure for a CK1 from Schizosaccharomyces pombe • Go to the Protein Data Bank (www.pdb.org) • Search for 1CSN • 1CSN is the pdb identifier for casein kinase - 1

5. Downloading the CK1 Structure Click “Download Files”and select “PDB File (Text)”

6. Dowloading CK1 structure • The file is downloaded into the “Downloads” folder • Pull up the Downloads window and drag the 1CSN.pdb file into the CK1-modeling folder you created.

7. Viewing the PDB File Text • Right click on the 1CSN.pdb file and choose “Open With…” • Click the radio button for “Select the program from a List” • Double click “Word” • You may want to adjust the margins to “narrow” under Page Layout to allow the file to be displayed well.

8. Anatomy of the PDB File • The Header

9. Anatomy of the PDB File • The amino acid sequence and secondary structure

10. Anatomy of the PDB File • The atoms, their amino acid, and their coordinates

11. Anatomy of the PDB File • Non-protein atoms like Mg, ATP, SO4, H2O, etc.

12. Anatomy of the PDB File • Connection table and end of file

13. Viewing Casein Kinase - 1 • Open the program VMD

14. Viewing Casein Kinase - 1 • You will see three windows

15. Viewing Casein Kinase - 1 • In the VMD Main window select“File → New Molecule…”

16. Viewing Casein Kinase - 1 • In the Molecule File Browser window that opens click “Browse” and find your pdb file (1CSN.pdb).

17. Viewing Casein Kinase - 1 • The “Filename” region should now have the path to your pdb file, and “Determine file type:” should have PDB • Click “Load”

18. Viewing Casein Kinase - 1 • The enzyme will appear in the OpenGL Display window. • Notice the various colors. What do they mean? • Left click on the black region and move your mouse around.

19. Viewing Casein Kinase - 1 • In the “VMD Main” window, click“Graphics → Representations”. • This will open a new window

20. Viewing Casein Kinase - 1 • Try changing the Drawing Method to see various representations like, CDW, CPK, Ribbons, new Ribbons, Cartoon, etc. • Now, set it to Ribbons

21. Viewing Casein Kinase - 1 • You’re now going to highlight some of the interesting features of CK1s • Click on Create Rep • In the Selected Atoms box, type “resname ATP” • Under the Draw style tab, set the Drawing Method to “Lines” and select “Color ID” under “Coloring Method”. This should highlight the ATP in a different color than the whole protein.

22. Viewing Casein Kinase - 1 • Highlight the ATP-binding pocket • Create Rep • Use the CPK drawing method and the Color ID coloring method to highlight residue 13 (this is Gly 83 in Yck2) • Create Rep • Use the CPK drawing method and the Color ID coloring method to highlight residue 15 (this is Gly 85 in Yck2). Keep the color the same for these amino acids because they’re both part of the ATP-binding pocket. • Repeat these steps for residue 18, 35, 148, 149, and 150.

23. Viewing Casein Kinase - 1 • Highlight the RD pocket • Create Rep • Use the CPK drawing method and the Color ID coloring method to highlight residue 124 (this is Arg194 in Yck2). Use a different color for the RD pocket than you used for the ATP-binding pocket. • Create Rep • Use the CPK drawing method and the Color ID coloring method to highlight residue 153 (this is Lys223 in Yck2). Keep the color the same for the amino acids that make up the RD pocket. • Repeat these steps for residue 170.

24. Viewing Casein Kinase - 1 • Highlight the activation loop • Click Create Rep • In the “Selected Atoms” box, type “residue 151 to 181” • This is the activation loop • Click “Create Rep” • On the “Draw style” tab select “ColorID” under “Coloring Method”. Choose a different color than you used for the RD pocket or the ATP-binding pocket.

25. Creating Representation • Here is a representation showing some important residues.

26. Measuring Distances • You can select atoms by changing the mouse behavior in the Mouse menu to “Label → Bonds” • Click on two atoms, and the distance between them will be shown

27. Measuring the ATP-binding pocket • You’ll use this feature to measure the distance between atoms that make up the ATP-binding pocket.

28. Saving Protein for Simulation • Open the Tk Console under the Extensions menu

29. Saving Protein for Simulation • In TkConsole, change directory to your modeling folder • It will depend on your computer folder hierarchy, but the command is something like • cd ../../../Users/Student/Documents/CK1-modeling • If you close VMD for any reason, you will need to re-enter this command to put yourself back in the proper working directory. • The default directory for VMD on Windows is C:/Program Files (x86)/University of Illinois/VMD if you mistakenly save work without changing directory first.

30. Saving Protein for Simulation • In TkConsole, set a variable “csn” that stores only the protein • set csn [atomselect top protein] • Write a new pdb file storing only the protein (no ATP, Mg, SO4, H2O will be saved) • $csnwritepdb csnp.pdb

31. Saving Protein for Simulation • You can select anything and everything you want rather than just the protein just like when you created representations. Some other options • all • protein • residue • resname • E.g., if you wanted to keep the ATP, you’d use the boolean operator OR • set csnatp [atomselect top protein OR resname ATP]

32. Check Your New csnp.pdb • Open with Word and make sure only amino acids 6-298 are in the file Beginning of file End of file

33. Open Your New pdb File • First, delete 1CSN by selecting it in the VMD Main window. • Next, select Delete Molecule from the Molecule menu. • Then, File → New Molecule…

34. Generate a psf file • The simulation software (namd ) requires a .psf • psf stands for protein structure file • Tells namd about • Charges and ionization state of histidine • The nature (stiffness) of bonds (C-C, C-N, C-O, etc.) • Non-bonded interactions • Angles (e.g., dihedral) • NAMD stands for NAnoscale Molecular Dynamics • It is pronounced NAM-DEE

35. Generate a psf file • Open the automatic psf builder • VMD Main • Extensions → Modeling → Automatic PSF Builder

36. Generate a psf file 1. Change the Output basename to csn 2. Leave the default topology 3. Click Load input files Just do this once! Don’t be impatient—you won’t see anything happen unless you can see the console window. 4. Click Guess and split… 5. Click Create chains. A message may pop up. This is fine. Click OK.

37. Check the TkConsole Output 1. You should see some warnings. These are OK as long as you see the two Info sections saying the psf and pdb were written. Wait until you see “Updating structures” and you are returned to a blinking cursor.

38. Check the Output Files Your modeling folder should have two files named csn. One is a .psf, and one is a .pdb. You can open the pdb in Word to make sure it is still amino acids 6-298 again.

39. Open Your New Files in VMD • First, delete csnp.pdband csn.psf by selecting them in the VMD Main window. • Next, select Delete Molecule from the Molecule menu. • Then, File → New Molecule… • Load csn.psf first, then load csn.pdb

40. Look at your New Files in VMD Display • When the psf was generated, VMD added hydrogen to all of the amino acids (white tips) • Hydrogens are not present in x-ray crystal structures because they are smaller (0.5 Å) than x-ray wavelengths (≥1 Å), and the single electron does not interact with x-rays

41. Make Your Mutation • Open the Mutator • VMD Main • Extensions → Modeling → Mutate Residue

42. Make Your Mutation If you correctly loaded psf and pdb files, these will be correctly filled in for you. Change the output name to csn_mutation. Enter the mutation target residue number. Enter the mutation you want. Remember, the numbering is different for this file: Yck2 S. pombe CK1 Mutated aa code Arg194 Arg130 GLN Lys223 Lys159 GLN Lys240 Lys176 GLN Ser243 Ser179 ASP Run mutator

43. Check Your Mutation • Open Tk Console and check that it completed successfully. • The mutation should have created a pdb and psf file. • Open the pdb file and make sure your mutation was successfully introduced. The file should be in your working directory (see slide 29).

44. Double mutations If you are doing a double mutation (e.g., R194Q/S243D), repeat the mutator steps to generate your second mutation, but use the mutated files with the single mutation as the starting point. Again, check your pdb file to make sure that both mutations you want are generated.

45. Solvating Your Protein • NAMD is an explicit solvent molecular dynamics simulator. This means it has water molecules surrounding the protein. Some simulators use implicit solvent that mimics water dielectric without the molecules being present in the calculations. Implicit solvents are faster to simulate but typically cause proteins to adopt more closed configurations. • Open the Solvator • VMD Main • Extensions → Modeling → Add Solvation Box

46. Solvating Your Protein If you correctly loaded psf and pdb files, these will be correctly filled in for you. Change Output: csn_mutation_wb Use Molecule Dimensions Click Solvate

47. Solvating Your Protein • VMD creates psf and pdb files with the water box (_wb). • Your new solvated protein should have appeared. • Delete the unsolvatedmolecule in VMD and create a nice representation of your solvated one.

48. Coordinates of Your Protein • We need to know where your protein in its water box sits in 3D space. Use TkConsole. • set everyone [atomselect top all] • measure minmax $everyone • Returns {xmin, ymin, zmin}{xmax, ymax, zmax} • Store these somewhere (in a lab notebook or in a file) • See image below for what the output looks like

49. Equilibrating Your Protein • Calculate the center of your water box. • Calculate the size of your water box

50. Equilibrating Your Protein • We need to get the protein to adjust itself because the mutation and water introduce new forces. In physics, forces have the potential to push things around. This is potential energy. NAMD will attempt to rearrange the protein to minimize the potential energy. This is the same as trying to find where all the forces exactly cancel one another. • Delete all open molecules using VMD Main. • Open your mutated .psf that has the water box, and without closing the open window, browse to and open your mutated .pdb that has the water box in the same window. • You can make a nice representationif you wish.