Premium
Development and Characterization of a Model Post‐Translationsally Modified Protein Library
Author(s) -
Major Emily K.,
Magnani Roberta,
Houtz Robert L.,
Bradley Luke H.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.791.17
Subject(s) - calmodulin , mutant , biology , linker , biochemistry , peptide library , target protein , lysine , calmodulin binding proteins , in vitro , computational biology , microbiology and biotechnology , amino acid , peptide sequence , enzyme , gene , operating system , computer science
From plants to humans, calmodulin lysine methyltransferase (CaM KMT) is a highly conserved protein responsible for the trimethylation of Lys‐115 in calmodulin (CaM). Recent findings suggest that CaM trimethylation is important in a wide range of metabolic, cellular, and developmental functions. Furthermore, we used CaM KMT as a protein engineering tool to post‐translationally modify a high‐quality protein library of the CaM central linker, using a coexpression system in E. coli . We showed that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM's thermal stability, and binding specificity towards CaM‐peptide target sequences. However, the substrate specificity of CaM KMT is unknown. A total of 40 conservative and non‐conservative amino acid substitutions were designed and introduced 3 residue positions (positions P(−3) to P(+3)) flanking the target Lys‐115 trimethylation site. In vitro trimethylation assays, using bacterially expressed and purified CaM KMT with purified mutants, identified positions and putative target sequences for CaM KMT. These results suggest alternate in vivo substrates and cellular roles for CaM KMT. Furthermore, these results provide further design information towards producing an unbiased and targeted post‐translationally modified library of novel sequences, thereby providing an additional tool for designing and generating proteins with stringent protein‐target specificities. Support or Funding Information NSF KY ESPCoR 0IA‐1355438 Bucks for Brains‐ University of Kentucky Office of Undergraduate Research This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .