The success of rational protein design can be enhanced by synthesizing and testing multiple designed proteins in the lab. We previously optimized binding of the MICA protein surface to its protein ligand, the homodimeric NKG2D immunoreceptor, with an iterative process resulting in seven MICA mutations that successfully enhanced the interaction. Here we apply these techniques to designing a single-chain form of NKG2D with an optimized homodimer interface.To effectively assess the binding of multiple designs, we assign multiple undergraduates to make the proteins in a biochemistry teaching lab course and to test them for binding in a survey of physical chemistry lab course, guiding and recording their progress with a set of GENI online tools we have developed. Surface Plasmon Resonance (SPR) binding analysis was made possible in this context with a BIAcore X instrument, which gave similar results as a BIAcore T100 instrument at <10% of the cost.Six linker designs were produced, purified, and tested, ranging from traditional gly-ser linkers to structure-based designs. The structure-based designs resulted in higher protein-protein affinities. Rosetta was used to redesign the NKG2D homodimer interface in two areas: by replacing a disulfide bond network at the top of the interface near the introduced linker, and in the hydrophobic center of the interface. Single-chain NKG2D versions with structure-based linkers containing optimized residues in each of these areas and in both areas together were produced and tested for binding. The redesigned NKG2D with the highest affinity will be used as the basis for future designs, such as optimization of the NKG2D interaction with MICA and studies of NKG2D-ligand specificity.[Partial support for this work was provided by the National Science Foundation's Transforming Undergraduate Education in Science, Technology, Engineering and Mathematics (TUES) program under Award No. 1322848.]

Rational protein design, SPR binding analysis, and iterative optimization of student-designed single-chain immunoreceptors

248th National Meeting (2014)

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