Incorporation of Non-Canonical Proline Residues into Proteins Expressed in Escherichia coli

Citation

Breunig, Stephanie Lynne (2023) Incorporation of Non-Canonical Proline Residues into Proteins Expressed in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8nj2-v152. https://resolver.caltech.edu/CaltechTHESIS:05222023-030948313

Abstract

Non-canonical proline residues expand the chemical space about proline, while maintaining some conformational properties of the canonical residue. The translational machinery of Escherichia coli can accommodate close structural analogs of proline, which has enabled the production of recombinant proteins that contain non-canonical residues at proline positions. However, proline mutagenesis in E. coli is restricted to a relatively small set of proline variants, and protein science and engineering efforts utilizing non-canonical proline residues are limited.

This thesis aims to expand the scope of proline analogs that can be accepted by E. coli, and demonstrate the utility of proline mutagenesis in modifying and studying protein behavior. In Chapter II, we describe the incorporation of three aliphatic proline residues into recombinantly-produced insulin, and find that these modest modifications at ProB28 alter the biophysical properties of the therapeutic protein. In particular, the addition of an exocyclic olefin at B28 accelerated insulin fibril formation, while 4-methyl substituents increased the rate of dissociation from the pharmaceutically-formulated insulin hexamer. We expand our proline mutagenesis approach to monomeric insulins in Chapter III. 4-fluorinated proline analogs replaced ProB29 of the fast-acting insulin lispro; 4S-fluorination of ProB29 slowed fibril formation. Chapter IV describes the incorporation of the photo-activatable proline analog "photo-proline" into proteins expressed in E. coli, and Chapter V discusses our efforts to engineer the E. coli prolyl-tRNA synthetase to accommodate more diverse proline substrates. Together, this work expands the proline analogs accessible to recombinant expression in E. coli, and demonstrates their use in probing and engineering the biophysical properties of proteins.

Thesis Files