Engineering of elastin using noncanonical amino acids Open Access

Kim, Wook Hyun (2007)

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Protein engineering has been widely used as a tool for globular protein design and modification, and also for engineering of protein-based materials. Conventional protein engineering approaches to protein-based materials have several limitations in that amino acid substitution in some cases alters the properties of the materials, and only the naturally occurring amino acids must be utilized. However, the use of noncanonical amino acids in protein engineering allows for maintaining the structure of the corresponding canonical amino acids, especially the imino acid proline, and introducing various functionalities into the side chain. Proline residues play important roles in protein structure and function. In order to more closely approximate the structural aspects of proline residue in a structurally important position, the substitution of proline with a proline analogue would be desirable for investigating contribution of proline to the conformational stability of the proteins.
The second chapter has focused on developing a series of proline auxotrophic E. coli expression strains that are competent for multi-site incorporation of a structurally diverse series of proline analogues into elastin-mimetic polypeptides and culture conditions that are compatible with high levels of analogue substitution within elastin-mimetic sequences. In order to achieve these, hyperosmotic culture media has been used in addition to overexpression of wild-type E. coli prolyl-tRNA synthetase and variants derived from site-directed mutagenesis of active site residues. The efficacy of co-translational incorporation judged by protein yield depended on the structural similarity between proline and proline analogues. The reliability of the methods was examined by quantitative and qualitative analyses of analogue substitution.
The rest of this work describes the effects of fluoroproline substitution on the biophysical properties of elastin-mimetic polypeptides, which may be attributed to the influence of pyrrolidine ring conformation on the stability of turn structure and the resulting self-assembly. Structural analyses of epimeric pairs of fluoroproline derivatives revealed that stereoelectronic and steric effects altered the main-chain dihedral angles and the ring pucker preference. Biophysical and computational studies of fluoro-elastins provided evidence that these two factors contribute to observed biophysical differences that arise from the presence of fluoroprolines at the structurally critical positions in the polypeptide sequence.

Table of Contents

Chapter 1. Introduction
     1. Proline-containing motifs
     2. Elastin-mimetic polypeptides
     3. Amino acid substitutions in engineering elastin
     4. Introduction of noncanonical amino acids as protein engineering method
Chapter 2. Co-translational incorporation of a structurally diverse series of proline analogues into elastin-mimetic polypeptides in an Escherichia coli expression system
     Experimental Section
           Materials and methods
           Physical and analytical measurements
           Plasmid construction
           Bacterial growth and expression
           Protein purification
     Results and Discussion
Chapter 3. A Stereoelectronic Effect on Turn Formation Due to Proline Substitution in Elastin-Mimetic Polypeptides
     Experimental Section
           Materials and methods
           Physical and analytical measurements
       Computational methods
     Results and Discussion
           Biosynthesis of elastin analogues
           Calorimetric measurement of the elastin phase transition
           Conformational analysis of elastin polypeptides
           Computational studies of (Pro-Gly) turns
Chapter 4. Fluoroproline Flip-Flop: Regiochemical Reversal of a Stereoelectronic Effect on Peptide and Protein Structure
     Experimental Section
           Materials and methods
           Physical and analytical measurements
           Chemical synthesis of model compounds (1) and (2) and free amino acids
           Protein expression and purification
     Results and Discussion
           Analysis of N-acetyl-(2R,3R)-3-fluoroproline methyl ester (1) and N-acetyl-(2R,3S)-3-fluoroproline methyl ester (2)
           Biosynthesis of elastin derivatives
           Conformational anaylsis of elastin polypeptides
           Calorimetric measurements of the elastin phase transition
Chapter 5. Conclusions

Appendix 1. CD and NMR spectra of other elastin analogues
Appendix 2. Morphology studies: Cryoetch-HRSEM and cryo-TEM images of elastin-1, elastin-2,and elastin-3
Appendix 3. Crystallographic data for N-acetyl-(2R,3R)-3-fluoroproline methyl ester and N-acetyl-(2R,3S)-3-fluoroproline methyl ester

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