The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 117-119). Also issued on the Internet.

published_or_final_version / Physiology / Master / Master of Medical Sciences

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.

Thesis (Ph. D. in Biological Science)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (M.S.) -- Mississippi State University. Dave C. Swalm School of Chemical Engineering. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 56-68).

Thesis (Ph.D. in Pharmacology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 145-170). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;