4 questions in protein engineering / biology

Three samples of tropomyosin fragments have been measured, one wild type and two mutants: Tpm WT, Tpm R91C and Tpm R91P. This fragment should form a coiled coil structure, therefore we expect to see spectra typical for alpha-helices. Our task is to find out if the mutations affect the structure and stability of this protein.

Wild type sequence:

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1.(15 points) Show sequences of all fragments aligned (R is the only residue to change; use courier font for alignment) with the heptad repeat (abcdefg, use as many repeats as necessary to align) and mark residues in a and d positions in the sequences as bold. First Leu (L) in sequences is in a position. Using ProtParam, calculate molecular mass and number of residues for each sequence, WT and the mutants.

2.(5 points) Find out what is position in the heptad repeat for the mutated Arg (R). Why may it be possible to affect the coiled coil structure by mutating this residue?

3. Use uploaded CD data measured for buffers (water in this case) and the protein fragments (dat files).

(a)( 5 points) Open data in Excel and create columns for protein spectra measured at 10oC (files Tpm312R91, cell2: WT, cell3:R91C, cell4:R91P) by subtracting CD values for buffers from CD values of proteins solutions. Each cuvette has its own CD spectrum, therefore use data for the same cell (cuvette) number when subtracting.

(b)(15 points) Create columns for mean residue ellipticity. Calculate mean residue ellipticity for each protein sample at 10oC before we started increasing temperature (concentrations are 0.23 mg/ml, 0.2 mg/ml and 0.34 mg/ml for Tpm WT, Tpm R91C and Tpm R91P, respectively; cuvette width is 0.1 cm). Find the equation for calculations in lecture notes.

(c)(20 points) Make one graph of mean residue ellipticities vs wavelength for all spectra of WT and the mutants at 10oC (before melting). Based on the spectra shapes, explain effect of mutations on the structure. What mutation has bigger effect?

(d)(15 points) Use data from mean residue ellipticities to calculate secondary structure content using BestSel website. Show results and compare with your conclusions based on spectra shapes.

(e)(5 points) What may be a reason for differences in results?

4.Use uploaded CD data for spectra at different temperatures, 10-70oC.

a)(5 points) Make a graph of dynode voltage vs wavelength for all data at 70oC. For each sample, find and show a wavelength region with data you cannot trust (if there is any).

b)( 10 points) For each sample make a graph of CD signal (in millidegrees, don’t need to calculate mean residue ellipticity but subtract corresponding buffer value) at 222 nm vs temperature. Note, that ellipticity of water does not change with temperature so you can subtract the same value from data at different temperatures but for the same cell number.

c) (5 points) What mutation has biggest effect on stability?

Submit Excel files with graphs and doc files with answers.

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