Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2012.
Heme c containing proteins are known for their intense colors and essential
functions in nature. These proteins contain heme that is covalently bound to the
protein. For part of the work in this thesis, we have developed fusion tags that contain
heme c, known as heme-tags, that reversibly bind an L-histidine immobilized
Sepharose (HIS) chromatography resin for affinity purification of recombinant
proteins expressed in Escherichia coli. The heme-tag HIS purification method
couples the ease of affinity purification with the convenience of visible detection for
protein tracking. In addition, we show that the heme-tag can be used to quantify the
protein.
Heme is covalently bound to native heme c proteins by several dedicated
biogenesis systems that exhibit a variable degree of diversity and substrate specificity.
Heme-tagged proteins are produced using a biogenesis system native to E. coli with
promiscuous substrate specificity. The biogenesis system that matures native
mitochondrial cytochromes c (cyt c) that function in cellular respiration and apoptosis
is referred to as cytochrome c heme lyase (CCHL). The substrate specificity of CCHL
has been explored, but the details are unclear. In this thesis, we show that CCHL can
mature the first 18 N-terminal residues of horse cyt c fused to a non-heme containing
protein, which demonstrates that the C-terminal portion of cyt c is unimportant for
substrate recognition. This work lends new insight into the substrate specificity of
CCHL and provides a new approach for producing artificial heme c proteins that
could find use in multiple applications.
In addition to developing novel biochemical applications of the heme tag, we
study the folding of the important model protein horse cyt c. Protein folding is
considered one of the most confounding problems in science, and cyt c has long been
a model protein to understand the general principles of folding. Herein, we show that
the folding of cyt c is more complex than previously reported using single molecule
fluorescence methods. We show evidence of multiple folding intermediates and
pathways as the protein folds on its energy landscape.