We are investigating coiled-coil protein structures to correlate the patterns of amino acid sequences and their stability. The coiled-coils are based on a repeating heptad structure with positions labeled A through G. Positions A and D are hydrophobic amino acids whose need to be buried drives the formation of the structure. Positions E and G are charged amino acids that stabilize the structure with electrostatic interactions on the sides of the coiled-coil. When all of the amino acid residues are coiled, the position G interacts with E from the other helix and the positions A and D and pack against the D and A from the other helix. Positions B, C, and F are turned away from the hydrophobic core meaning they do not interact with other positions. We are specifically looking at the coiled-coils from GrpE homologues from T. thermophilus and E. coli, which have very different stabilities as a result of the native environments of the organisms. T. thermophilus GrpE denatures at a much higher temperature than the E. coli GrpE, but different portions of these proteins denature first. We will measure the strength of the interactions between helices using self-quenching fluorophores where the high-intensity state corresponds to the denatured coiled-coil with an increase in the distance between fluorophores allowing them to fluoresce. The low-intensity state corresponds to our coiled-coils folded state with the fluorophores close together and quenched. We will be able to graph these values and find at what concentration of Guanidine HCl or at what temperature do the coiled-coils denature. This research will compare the stabilities of the coiled-coil regions and will provide the opportunity to study how their protein sequence relates to their thermal properties by mutating the sequences to strengthen or weaken the inter-helix interactions and measuring their new melting temperatures.

Correlating the stability of coiled-coil structures with changes in the sequence

255th National Meeting (2018)

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