Question

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: GSHMCGGLKDAQEKLELAEKKAADAEAEVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAA DESERGMKVIENRALKDEEKMELQEIQLKEAKHI 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?

Answer 1

Question 1

Tpm WT (Amino Acid Sequence)

GSHMCGGLKD AQEKLELAEK KAADAEAEVA SLNRRIQLVE EELDRAQERL ATALQKLEEA
       
EKAADESERG MKVIENRALK DEEKMELQEI QLKEAKHI

Molecular Mass of Tpm WT (Using ProtParam)= 11055.48 Dalton / 11.5 Kda

Number of residues = 98

Tpm R91C

GSHMCGGLKD AQEKLELAEK KAADAEAEVA SLNRCIQLVE EELDRAQERL ATALQKLEEA
       
EKAADESERG MKVIENRALK DEEKMELQEI QLKEAKHI

Molecular Mass of Tpm R91C (Using ProtParam)= 11002.43 Dalton / 11 Kda

Number of residues = 98

Tpm R91P

GSHMCGGLKD AQEKLELAEK KAADAEAEVA SLNRPIQLVE EELDRAQERL ATALQKLEEA
       
EKAADESERG MKVIENRALK DEEKMELQEI QLKEAKHI

Molecular Mass of Tpm R91P (Using ProtParam)= 10996.41 Dalton / 10.9 Kda

Number of residues = 98

Question 2

It affect the coiled structure by mutating R residue. Please see the all three gif files.  

For Tpm WT
Coils output for UNKNOWN window=14 window=21 window=28 0.8 0.6 F 0.4 0.2 0 10 20 30 40 50 60 70 80 90 100

For Tpm R91C

Coils output for UNKNOWN window=14 window=21 window=28 0.8 0.6 F 0.4 0.2 0 10 20 30 40 50 60 70 80 90 100

For Tpm R91P

Coils output for UNKNOWN window=14 window=21 window=28 0.8 0.6 F 0.4 0.2 0 10 20 30 40 50 60 70 80 90 100