The experimental results reported here test the hypothesis that incorporation of amino acid analogs into periodic proteins at multiple positions should yield new materials with unique physicochemical properties associated with the amino acid analogs. Two amino acid analogs with trifluoromethyl and thiophene side groups were featured in this study, i.e., trifluoroleucine and 3-thienylalanine. A coupled in vitro transcription-translation assay procedure was first established to assess the ability of the Escherichia coli protein synthesis machinery to use non-natural amino acids. Second, a general strategy for incorporating amino acid analogs in vivo was developed. This procedure uses a bacteriophage T7 expression system and high cell density fermentation. The method was used to make tens of milligrams of periodic proteins of the general sequence $\{$(GlyAla)$\sb3$GlyXxx$\}\sb{\rm n}$ where Xxx is either leucine, trifluoroleucine, phenylalanine or 3-thienylalanine. The proteins were designed to adopt a "lamellar" morphology in the solid state on crystallization. Incorporation of trifluoroleucine into the recombinant protein was established by elemental analysis, NMR analysis and mass spectroscopy. Spectroscopic analysis by $\sp{19}$F NMR showed that both 2S isomers of trifluoroleucine are used in protein synthesis. Periodic proteins containing leucine and the trifluoroleucine were crystallized. Structural analysis based on X-ray diffraction and infrared spectroscopy indicated that the chains form predominantly beta sheets with a lamellar morphology. Contact angle measurements indicated that the fluorinated protein had a lower surface energy compared with the non-fluorinated counterpart. These results demonstrate that protein materials with modified surface properties can be created by incorporating multiple trifluoromethyl groups at precise positions. A periodic protein containing 3-thienylalanine in place of phenylalanine was also prepared. Results from elemental and amino acid analyses, and NMR and UV spectroscopy indicated that the 3-thienylalanine was substituted at least 80% in the bulk sample and was not modified on incorporation. The 3-alkylthiophene side chain of 3-thienylalanine should be susceptible to electrochemical polymerization, opening a route to genetically engineered polymeric materials with useful electronic properties. Taken together, the results of this investigation establish the feasibility of preparing recombinant periodic proteins in vivo that contain useful amino acid analogs at multiple positions. The general strategy used should be useful in designing and fabricating additional protein-based materials with unique physicochemical properties.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-9039 |
| Date | 01 January 1995 |
| Creators | Kothakota, Srinivas |
| 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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