Through the design of chimeric proteins, diverse functionality may be incorporated into a single molecular entity, improving the utility of the whole over that of the individual component parts. One way of achieving a protein chimera is through the addition of a flexible peptide loop such as an antibody binding loop or an enzyme recognition sequence into the structure of an unrelated protein, providing novel functionality at the site of insertion. Traditionally, existing loop regions or the N- and C-termini of a target protein are chosen for the addition of novel peptide sequences in generating these types of chimeric proteins; however, restricting design to these sites is extremely limiting as the terminal residues and flexible loops of the target protein may be required for proper function should they be available for modification at all. To overcome this limitation in chimeric protein design, we are developing structural screening techniques for identifying sites within a target protein that can accommodate the insertion of peptide loops without negatively impacting protein function. In particular, we have created a library of constructs containing a 13-residue peptide loop (know as the LAP sequence) cloned into the enzyme lipase A from Bacillus subtilis at numerous unique sites within diverse secondary structural elements and with varying solvent accessibilities, local charge environments, and B-factors. We explore correlations between these local structural parameters and the expression level, activity retention, and thermal stability of the constructs in order to establish or rule out structural predictors for successful insertion sites. Additionally, we compare this dataset to scores obtained for each construct when modeled with the kinematic closure loop modeling application of the Rosetta protein modeling software suite. Our results indicate that unintuitive sites in lipase (including sites within alpha helices) can accommodate LAP insertion in such a way as to minimally impact overall protein expression and function and that Rosetta scores may be used to reduce the number of constructs generated and tested in finding these sites. We also propose a general strategy for screening protein structures for possible loop insertion sites that can be accomplished with limited computational power. Overall, our results suggest a general structure-based approach for reducing the search space in designing protein chimeras that contain peptide loops.

Structurally guided design of chimeric proteins containing novel peptide loops

253rd National Meeting (2017)

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