Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Plant Pathology and Crop Physiology

First Advisor

Norimoto Murai

Abstract

Common beans are widely utilized as a food source, yet are limited as a complete source of protein due to low levels of methionine, an essential amino acid for humans. A protein engineering strategy was developed to increase the methionine content of phaseolin, the primary seed storage protein in common beans. The engineering strategy consists of three major parts. In the first part, a set of biophysical probes was developed to characterize the stability of wild-type and modified phaseolin proteins. I used absorbance, fluorescence emission, circular dichroism, and fluorescence polarization anisotropy to monitor phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature. The protein denatured irreversibly at 65$\sp\circ$C when dissolved in 6.0 M guanidinium chloride, indicating that phaseolin has exceptional structural stability. In the second part, the complete three-dimensional structure of phaseolin was generated from $\alpha$-carbon coordinates. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. Three types of modifications were tested: replacement of 10 variant hydrophobic residues with methionine in each of the $\beta$-barrel structures, insertion of short methionine-rich sequences at surface exposed regions of the protein, and insertion of a 9 kd methionine-rich domain into the N-terminal hypervariable region of phaseolin. In the last part, 24 mutant phaseolin cDNAs were constructed and expressed in E. coli to determine the effects of the mutations on the protein structure in vivo. Methionine enhancement ranged from 5 to 45 residues. Thermal denaturation of purified proteins demonstrated that significant modifications for methionine enhancement in the $\beta$-barrels did not alter the structural stability of the protein. In addition, protein denaturation was a reversible event, which allowed a thermodynamic analysis of protein stability. The utilization of these strategies permits a thorough investigation of the effects of mutagenesis on phaseolin stability. Since mutant proteins with properties most similar to wild-type are likely to survive the process of accumulation in seeds, this knowledge is crucial for the identification of optimal candidates for plant transformation.

Pages

229

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