Publication Highlights

Deep sequencing methods for protein engineering and design

This reviews the pairing of next generation sequencing technologies with high throughput screens for studying and designing protein-protein interactions and enzymatic functions.

Wrenbeck EE, Faber M, Whitehead TA (2017) “Deep sequencing methods for protein engineering and design”, Current Opinion in Structural Biology 45:36-44

Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing

By combining fluorescence activated cell sorting, yeast surface display, and next generation sequencing we present and validate an experimental pipeline that determines conformational epitopes. We determine the epitopes for multiple antigen-antibody interactions and compare our findings with previously reported X-ray crystallography data.

Kowalsky CA, Faber M, Nath A, Dann H, Kelly VW, Liu L, Shanker P, Wagner EK, Maynard J, Chan C, Whitehead TA (2015) “Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing”, Journal of Biological Chemistry 290 (44), 26457-26470 doi:10.1074/jbc.M115.676635

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

Cellulases bind non-specifically to lignin, making economical lignocellulusic-mediated conversion more difficult. The mechanisms behind this adsorption are unclear, We use computational design to systematically investigate the role of protein surface potential on lignin adsorption. We designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge and total charge density typical for natural proteins. Low net negative charge was the single largest predictor for low lignin-protein adsorption, and we are using this insight to redesign cellulases that are stable in the presence of lignin.

Haarmeyer C, Smith MD, Chundawat S, Sammond D, Whitehead TA, (2016) “Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein”, Biotechnology & Bioengineering in press

Plasmid-based one-pot saturation mutagenesis

 In this paper we present a new method, Nicking Mutagenesis, to generate comprehensive, single-site saturation mutagenesis libraries in a single day in a single tube using plasmid dsDNA as the substrate. The method employs complementary nicking endonucleases, exonucleases, and thermostable high-fidelity DNA polymerases and ligases to generate complex libraries all on a thermocycler. To improve accessibility of the method, a positive control plasmid for running the mutagenesis can be obtained from Addgene.com (Plasmid #80085) and detailed protocols can be found on the Nature Protocol Exchange (doi:10.1038/protex.2016.061).

Wrenbeck, E. E., Klesmith, J. R., Stapleton, J. A., Adeniran, A., Tyo, K. E., & Whitehead, T. A. (2016). Plasmid-based one-pot saturation mutagenesis. Nature Methods, 13(11), 928-930.

Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning.

Enzymes find utility as therapeutics and for the production of specialty chemicals. Changing the amino acid sequence of an enzyme can increase solubility, but many such mutations disrupt catalytic activity. To evaluate this trade-off, we developed an experimental system to evaluate the relative solubility for nearly all possible single point mutants for two model enzymes. We find that the tendency for a given solubility-enhancing mutation to disrupt catalytic activity depends, among other factors, on how far the position is from the catalytic active site and whether that mutation has been sampled during evolution. We develop predictive models to identify mutations that enhance solubility without disrupting activity with an accuracy of 90%. These results have biotechnological applications.

Klesmith, J. R., Bacik, J. P., Wrenbeck, E. E., Michalczyk, R., & Whitehead, T. A. (2017). Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. Proceedings of the National Academy of Sciences, 114(9), 2265-2270.