University of Kentucky

Poster Title

An Investigation into the Relationship Between Calmodulin Protein Conformation and Binding Specificity

Institution

University of Kentucky

Abstract

A multitude of cellular processes, ranging from cell survival to neuronal excitability, are regulated by proteins that bind to a large number of diverse and physiologically vital binding partners. For nearly 30 years, calmodulin (CaM), the central calcium signaling transducer, has been a model system to study pervasive binding specificity. However, after numerous structural and biochemical studies, the molecular basis of this binding activity remains unclear. Given that CaM binding affinity arises from a combination of amino acid contacts, protein combinatorial libraries offer an attractive approach towards investigating CaM binding specificity by identifying members with altered binding specificity for downstream biochemical structure/function studies. To maximize this search, productive regions of protein sequence space (i.e. folded and soluble) must be sampled. Here we present the first application of the binary patterning approach of combinatorial protein library design to the CaM central linker region. This high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. In addition, ANS-binding data showed that each of these possesses significant diversity in binding specificity. Collectively, this data support that the binary patterning approach, when applied to the highly conserved CaM protein, can yield large collections of folded, soluble and highlyexpressible proteins, which will facilitate the investigation of selected proteins with altered CaM binding specificity. Furthermore, this research introduces a new platform biotechnology for downstream use in the state’s agricultural, pharmaceutical, and biotech industries.

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An Investigation into the Relationship Between Calmodulin Protein Conformation and Binding Specificity

A multitude of cellular processes, ranging from cell survival to neuronal excitability, are regulated by proteins that bind to a large number of diverse and physiologically vital binding partners. For nearly 30 years, calmodulin (CaM), the central calcium signaling transducer, has been a model system to study pervasive binding specificity. However, after numerous structural and biochemical studies, the molecular basis of this binding activity remains unclear. Given that CaM binding affinity arises from a combination of amino acid contacts, protein combinatorial libraries offer an attractive approach towards investigating CaM binding specificity by identifying members with altered binding specificity for downstream biochemical structure/function studies. To maximize this search, productive regions of protein sequence space (i.e. folded and soluble) must be sampled. Here we present the first application of the binary patterning approach of combinatorial protein library design to the CaM central linker region. This high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. In addition, ANS-binding data showed that each of these possesses significant diversity in binding specificity. Collectively, this data support that the binary patterning approach, when applied to the highly conserved CaM protein, can yield large collections of folded, soluble and highlyexpressible proteins, which will facilitate the investigation of selected proteins with altered CaM binding specificity. Furthermore, this research introduces a new platform biotechnology for downstream use in the state’s agricultural, pharmaceutical, and biotech industries.