NSF EPSCoR: Library Strategy for Determining Post-Translational Modification Specificity

Grants and Contracts Details

Description

The central dogma of biology defines the role of genes as the codes that make proteins and their functions possible. Protein engineering is a growing sector of biotechnology and medicine. The biopharmaceutical industry depends on protein engineering to produce over a quarter of all emerging drugs. The demand for these engineered molecules to specifically perform their designed functions (without side effects) increases significantly. While combinatorial libraries have been utilized as powerful technology for the development of novel protein specificity, post-translational modifications, which nature uses to alter protein activity, are overlooked. To incorporate these regulatory elements into protein combinatorial libraries, we previously developed a bacterial co-expression system, utilizing calmodulin methyltransferase (CaM KMT) as a model system, to completely trimethylate a diverse protein library of the calmodulin (CaM) central linker region. Characterization of 17 randomly selected library members show that all library sequences were over-expressed and post-translationally modified. In addition, we show 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, to guide future library designs and applications of this technology, it is necessary to gain a better understand the binding specificity of CaM KMT. We propose to construct and characterize 42 different mutations, using a library assembly approach, designed to alter/disrupt the charges around the CaM KMT target and solvent-accessible residue, Lysine-115. Collectively, these data and ongoing studies will identify the putative CaM KMT recognition sequence for identifying other potential cellular targets of this enzyme, as well as a tool for future library designs.
StatusFinished
Effective start/end date9/18/177/31/18

Funding

  • National Science Foundation

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