Design of photoisomerizable amino acids and their incorporation into biological peptides and enzymes.
Published: 2004
Language: eng
Number of pages: 243
Description: This thesis describes the development and design of photoisomerizable peptides and proteins. Enzymes containing photoisomerizable amino acids or peptide segments could be used to study cellular processes, control or modify enzymatic cascade systems, or switch substrate specificity in a catalytic system.A family of photoisomerizable amino acids based on the photochromophore azobenzene were synthesized: phenylazophenylalanine (PAP), phenylazo-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (PATIC), p-carboxy-phenylazophenylalanine (CPAP), and  p-carboxyphenylazo-C-ethylamide phenylalanine (CPAPea). The amino acids were designed with N-alpha-fluoroxymethyl (Fmoc) protection to facilitate Fmoc-based solid phase synthesis.The photoisomerizable S'-peptides were used to develop photo-regulated RNase S analogues. We performed kinetic analyses of RNase S analogues containing PAP in positions 4, 7, 8, 10, 11, and 13, PATIC in position 11, and CPAP in positions 2 and 11. For an enzyme containing a single photoisomerizable residue, the maximum extent of photo-conversion (approximately 96% trans 14% cis for the dark-adapted system and 10% trans/90% cis under irradiated conditions) sets a limit on the maximum change in initial rate of &sim;25-fold, if the cis form is the more active isomer, and &sim;10-fold if the trans form is more active. The results suggest that photoisomerization could substantially alter enzyme kinetic constants, but that a different approach might be required for realizing maximal photo-control in such systems.Photoisomerizable residues were incorporated into a number of polypeptide sequences including the RNase S-peptide sequence (S'-peptides), a peptide with strong helical propensity (the DAJ1 peptide), and a peptide based on a mini-protein motif (the BBAzo peptide). The properties of the photoisomerizable residues (PAP, PATIC, CPAP, and CPAPea) were characterized following incorporation into polypeptide sequences to determine physical properties of the residues in an aqueous environment. Structural investigations were carried out on photoisomerizable peptides using circular dichroism spectroscopy, molecular modeling and two dimensional NMR. The effect of photoisomerization on the helix content of modified S-peptides and the DAJ1 peptide were analyzed and changes in structural stability were studied in the photoisomerizable BBAzo peptide.
Notes: Adviser:  G. A. Woolley.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2398.