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PROJECT SUMMARY/ABSTRACT
After proteins are synthesized on the ribosome, their activity, stability, and localization are often
regulated by post-translational modification (PTM) enzymes. Such modifications are essential for cellular
function in all domains of life, and dysregulation of PTM processes is linked to myriad adverse medical
conditions, including cancer and heart disease. A long-term goal of this work is to understand how PTM
enzymes select their targets, particularly where recognition elements on the target protein are distant
from actual sites of modification—a phenomenon termed “alloselectivity.” A second long-term objective
is to establish a general system for engineering proteins and peptides to be modified with user-defined
PTMs. The proposed work uses the micrococcin biosynthetic pathway as a model analytical system.
Micrococcin is an antimicrobial compound resulting from over 20 post-translational chemical
conversions on a 14-amino acid precursor peptide substrate. The subjects of this study are the
micrococcin biosynthetic proteins TclE, TclI, TclJ, and TclN. TclE is the substrate peptide, TclJ and TclN
are modification enzymes, and TclI is the coupling protein that recruits TclJ/N to the TclE substrate.
Proposed experiments probe the structural features underpinning this PTM complex, with an emphasis
on the role of TclI as a crucial structural hub that physically joins the other three components. This four-
protein complex can be functionally expressed at high levels in E. coli cells. The specific aims of the
project are to (i) characterize substrate (TclE) recognition by TclI using a series of mutations that are
guided by structural predictions, (ii) characterize the stable interactions between TclI and the TclJ/N
enzymes, using a combination of mutational and analytical biochemical approaches, and (iii) design,
build, and test prototypes of engineered PTM complexes that use elements of TclI and TclE to couple
heterologous protein kinase domains to engineered peptide substrates. This work will be carried out
primarily by an interdisciplinary team of undergraduate researchers. They will employ methods that
include plasmid manipulation, site-directed mutagenesis, in vivo bacterial two-hybrid analyses,
chromatographic co-purification, chemical cross-linking coupled with mass spectrometric analysis, and
computational protein structural modelling.
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