Date of Award

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biotechnology Science and Engineering

Committee Chair

Robert L. McFeeters

Committee Member

Leighton Coates

Committee Member

William N. Setzer

Committee Member

Joseph Ng

Committee Member

Krishnan Chittur

Subject(s)

Phenylalanine., Proteins--Structure., Biomolecules., Genetics.

Abstract

Macromolecular complexes are at the forefront of structural biology. Despite technological advances, there remains a significant size barrier for study of these systems. Site specific labeling of ILV methyl residues has been used to study systems such as the proteasome, but other methods are still needed. Aromatic amino acids have many of the advantages of methyl containing residues and are desirable for site specific labeling. They are often found in the protein core and at important interaction interfaces. Aromatic side chains have positions with multiple magnetically equivalent 1H probes and distinct carbon chemical shifts. Side chain ring flipping results in reduced effective correlation times. Thus aromatic side chains have potential to be used as excellent probes for large macromolecule systems. Using metabolic engineering, a bacterial system was constructed that produces phenylalanine with 13C carbons exclusively at the fÑ, f×, and fÕ positions of a phenylalanine residue (Figure 1.) This labeling pattern of every other carbon labeled eliminates j-coupling, increasing the signal and results in cleaner spectra. Once the pattern specifically labeled phenylalanine was produced and purified, phenylalanine was incorporated into recombinant ubiquitin with high (>90%) efficiency and minimal isotope scrambling. This was accomplished by the addition of a shikimate inhibitor to shut down the natural synthesis of aromatics in combination with mass action to push back on the most scrambled residues. This method has been applied to two proteins varying in size and aromatic amino acid composition to provide a general protocol amenable to any system. With incorporation of pattern specifically labeled phenylalanine into an unknown complex in mind, Small Angle Neutron Scattering data were obtained on the Pth1: peptidyl-tRNA complex to determine the overall shape of the complex. Now that the orientation of the components of the complex are known, it is possible to complement this data with high resolution NMR utilizing pattern specifically labeled residues to determine how this enzyme interacts with its natural substrate. Figure 1: Pattern Specifically Labeled phenylalanine. Labeled carbons indicated by Greek letters.

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