Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Alan Voskamp Klotz


A novel post-translationally modified asparagine, $\gamma$-N-methylasparagine (NMA), has been characterized at the $\beta$-72 site in many phycobiliproteins. The behavior of a NMA-containing peptide under Edman degradation conditions employed for sequence analysis was examined; NMA can be reproducibly identified in protein sequences by careful attention to repetitive yields and the presence of minor peaks. The effects of asparagine methylation on photosynthetic rates in the cyanobacterium Synechococcus PCC 7942 and two methylase-minus mutants was measured by steady-state oxygen evolution in whole cells. The methylase-minus mutants demonstrated lower rates of electron transfer through Photosystem II under conditions in which phycobilisomes were preferentially illuminated at low light intensity. Asparagine methylation is also associated with a selective growth advantage upon cells containing the modification. When grown under low light illumination for 200 generations, cells containing NMA outcompete the unmethylated strain at a rate of 0.38% per generation. Thus a plausible selective advantage for methylation can be demonstrated. Two site-specific mutants of C-phycocyanin from Synechococcus PCC 7002 in which $\beta$-72 NMA has been replaced with either aspartate or glutamine have been extensively characterized. The presence of NMA improves Photosystem II electron transfer under broadband and specific illumination conditions in whole cells. Replacements of NMA at $\beta$-72 of C-phycocyanin cause blue-shifts in the absorbance spectra and a decrease of approximately 15% in fluorescence quantum yields. Fluorescence studies have revealed that NMA is associated with improved energy transfer efficiency in both C-phycocyanin and isolated phycobilisomes that is accomplished by a decrease in non-radiative pathways of deexcitation other than resonance energy transfer. Molecular dynamics calculations are consistent and suggest that NMA alters hydrogen bonding networks and chromophore geometry. The effects of NMA replacement on C-phycocyanin stability have been measured by urea and thermal denaturation. While $\beta$-72 substitution does not affect overall free energies of folding, the replacements seem to alter the denatured state of the protein and quaternary structure dissociation.