Genetic engineering of protein-based polymers offers distinct advantages over conventional synthesis of polymers. Microorganisms can synthesize high molecular weight materials, in relatively large quantities, that are inherently stereoregular, monodisperse, and of controlled sequence. In addition, specific secondary and higher order structures are determined by this protein sequence. As a result, scientists can design polymers to have unique structural features found in natural protein materials and functional properties that are inherent in certain peptide sequences. For this reason, genetic engineering principles were used to create a set of artificial genes that encode twelve macromolecules having both $\alpha$-helical and disordered coil protein sequences with the last amino acid being cysteine (cys) or tryptophan (trp). Triblock copolymer sequences having coiled-coil protein ends, A or B, where A and B represent $\alpha$-helical acidic and basic leucine zipper proteins, separated by a water soluble flexible spacer coil protein, C, where C represents ((AG)$\sb3$PEG) $\sb{\rm n}$ (n = 10 or 28), showed reversible physical gelation behavior. This behavior is believed to result from the aggregation of two or more helices that form physical crosslinks with the disordered coil domain retaining solvent and preventing precipitation of the chain. Diffising wave spectroscopy was used to investigate the gelation behavior of AC$\sb{10}$Acys in buffer when environmental conditions such as pH, temperature, and concentration were varied. The dynamic intensity autocorrelation function recorded over time for 5% (w/v) AC$\sb{10}$Acys showed that the protein behaved as a gel at pH 6.7-8.0 and that the melting point was between 40$\sp\circ$C and 48$\sp\circ$C. In addition to the triblock results, the incorporation of 5$\sp\prime$,5$\sp\prime$,5$\sp\prime$-trifluoroleucine (Tfl) in place of leucine (Leu) in the A and B blocks was accomplished by synthesizing proteins in bacterial hosts auxotrophic for Leu. The substitution of Tfl for Leu in A and B was confirmed by electrospray mass spectrometry. Amino acid analyses performed on purified Tfl A and Tfl B populations suggested 66% and 38% levels of Tfl substitution, respectively. Thermal denaturation temperatures measured by circular dichroism of the Tfl containing helices were higher than those of the corresponding Leu containing helices by 8$\sp\circ$C and 13$\sp\circ$C for A and B respectively.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-2953 |
| Date | 01 January 1997 |
| Creators | Petka, Wendy Ann |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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