Biochem assignment 2

BIOC 405 Assignment 1: Dr Moore

Due Friday March 1st, 2019 before 16:00 in Room 3D30.8 HSc

1. (a)In your handout for protein kinase A, there is a table of known substrate sequences, in other words sequences of peptides phosphorylated by PKA. Please do your best to align the substrate sequences provided, and from the alignment, predict what a good consensus substrate for PKA will be. To present your alignment, please use an equal width font for the protein sequences (Courier or Courier New work well). Highlight the P(0) residue, P(-1) etc. (b)The regulatory subunit (R) of protein kinase A has a short sequence (RRRRGAISA that is critical for inhibiting the activity of the kinase catalytic subunit. This short sequence of the R-subunit actually sits in the active site cleft of PKA in the crystallographically-determined structure of the inhibited RC complex. Can you deduce what the function of this sequence is? Using the answer to part (a) as a guide, please align the inhibitory sequence with the known substrate sequences to deduce how this sequence likely functions to inhibit PKA. Furthermore, using your class notes, can you make a guess at which residues on PKA might interact with specific residues from the R-inhibitory peptide? 2. For a regularly spaced 1-Dimensional array of atoms (spacing =13 Å) calculate the total number of diffraction maxima and their scattering angles (for perfect in phase scattering from the atoms in the 1-D array) between scattering angles of zero and ninety degrees Use a wavelength of d=1.25 Å. Please include a drawing to explain the diffraction condition and show your calculations.

3. Using site specific mutagenesis to change residues in the substrate binding cleft of PKA (not residues involved in catalytic roles), how would you alter PKA’s substrate specificity at P-3, and P-2 to Glu and P+1 to Asn? By this, I mean how would you specifically make mutations in the PKA enzyme amino acid sequence (not the substrate sequence) that would select for binding and phosphorylation of a peptide sequence that would clearly differ from the known substrate sequence preferred by PKA as outlined above. Be sure to clearly highlight exactly what residues in the PKA sequence you would have to change (and to what amino acid) to achieve this.

4. The following lines of data describe the atomic coordinates for an arginine residue in a protein molecule in PDB (protein data bank) format. Since proteins are three-dimensional objects, the position of each atom is specified in space by its X, Y and Z coordinates. On each line of a PDB formatted file, the atom number is given, the atom type is next (e.g. N- backbone nitrogen, backbone carbonyl oxygen etc), the residue name (here ARG 431 in chain D; in this instance the protein crystal contains four independent copies of the polypeptide chain, labelled A through D), then the X-coordinate for that atom, the Y-coordinate for that atom and the Z-coordinate for that atom (given in Ångstroms = 10-10 m), a number called the occupancy (here 1.0) and another number the B-factor that describes the average motion of the atom in the protein structure (units in squared Ångstroms or Å2).

D21 = {(X2-X1)

2 + (Y2-Y1) 2 + Z2-Z1)

2}1/2 Atom Residue X Y Z Occ B-factor ATOM 2114 N ARG D 431 52.470 15.386 15.252 1.00 14.20 ATOM 2115 CA ARG D 431 51.319 16.197 15.632 1.00 15.27 ATOM 2116 CB ARG D 431 50.056 15.350 15.651 1.00 15.67

ATOM 2117 CG ARG D 431 49.597 14.728 14.328 1.00 18.26 ATOM 2118 CD ARG D 431 48.314 13.894 14.514 1.00 21.61 ATOM 2119 NE ARG D 431 47.564 13.503 13.331 1.00 24.52 ATOM 2120 CZ ARG D 431 47.246 14.316 12.351 1.00 25.92 ATOM 2121 NH1 ARG D 431 47.643 15.558 12.409 1.00 27.74 ATOM 2122 NH2 ARG D 431 46.562 13.879 11.299 1.00 26.98 ATOM 2123 C ARG D 431 51.537 16.895 16.989 1.00 15.54 ATOM 2124 O ARG D 431 50.664 17.535 17.502 1.00 15.87 Using the coordinates provided, explicitly draw the structure of CD, NE, CZ, NH1 and NH2 atoms of the side chain. Then calculate the bond lengths for the CZ-NH1 and the CZ-NH2 and CZ-NE bonds of the side chain using the given atomic positions and the provided distance formula. Then draw the chemical structure (as you would in organic chemistry class) of the guanidinium group of the arginine side chain and draw any allowable resonance structures if you think any of the bonds are delocalized. Also show the likely positions of the hydrogen atoms. Hint, the bond lengths of the C-N bonds should tell you if resonance is occurring (a single CN bond is about 1.46 Angstroms in length, and a double CN bond is about 1.22 Å in length). 5. Using your knowledge of side chain torsion angles and the results of question 4, show what are the rotatable bonds for the arginine side chain. Then please draw carefully (using a Newman projection as demonstrated in your class notes) the low-energy conformations about the Chi2 and Chi4 angles of arginine. Also, describe the hybridization of the atoms on either side of the Chi2 and Chi4 bonds. Please explain your results with respect to what we covered in class.