Poster Title

Development of a Model Post-Translationally Modified Protein Library

Grade Level at Time of Presentation

Junior

Institution

University of Kentucky

KY House District #

72

KY Senate District #

27

Department

Anatomy and Neurobiology

Abstract

Development of a Model Post-Translationally Modified Protein Library

Emily Major1*, S Maxwell Scalf1*, Katie Kloska1*, Lydia Fletcher1*, Roberta Magnani2, Robert Houtz2, Luke H. Bradley1,3

  • * Contributed equally to the project and presentation

  1. Department of Anatomy & Neurobiology, University of Kentucky College of Medicine

  2. Department of Horticulture, University of Kentucky College of Agriculture

  3. Department of Molecular & Cellular Biochemistry, University of Kentucky College of Medicine

As the use of proteins has become widespread in biotechnology and medicine (> $60B/year industry world-wide), the demand for these engineered molecules to specifically perform their desired/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 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 the protein associated with the binding of calcium, the protein’s thermal stability, and binding specificity towards CaM-peptide binding 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 constructed and verified 22 different mutations designed to alter/disrupt the charges around the CaM KMT target and solvent-accessible residue, Lysine-115. Collectively, these data and ongoing studies suggest that the use of CaM KMT to produce an unbiased and targeted post-translationally modified library of novel sequences is possible, thereby providing an additional tool for designing and generating protein with stringent protein-target specificities for biomedicine.

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Development of a Model Post-Translationally Modified Protein Library

Development of a Model Post-Translationally Modified Protein Library

Emily Major1*, S Maxwell Scalf1*, Katie Kloska1*, Lydia Fletcher1*, Roberta Magnani2, Robert Houtz2, Luke H. Bradley1,3

  • * Contributed equally to the project and presentation

  1. Department of Anatomy & Neurobiology, University of Kentucky College of Medicine

  2. Department of Horticulture, University of Kentucky College of Agriculture

  3. Department of Molecular & Cellular Biochemistry, University of Kentucky College of Medicine

As the use of proteins has become widespread in biotechnology and medicine (> $60B/year industry world-wide), the demand for these engineered molecules to specifically perform their desired/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 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 the protein associated with the binding of calcium, the protein’s thermal stability, and binding specificity towards CaM-peptide binding 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 constructed and verified 22 different mutations designed to alter/disrupt the charges around the CaM KMT target and solvent-accessible residue, Lysine-115. Collectively, these data and ongoing studies suggest that the use of CaM KMT to produce an unbiased and targeted post-translationally modified library of novel sequences is possible, thereby providing an additional tool for designing and generating protein with stringent protein-target specificities for biomedicine.