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YSD: Engineering Molecular Interactions

YSD: Engineering Molecular Interactions. Target protein is on the surface – biophysical characterization of binding by flow cytometry High-throughput & versatile molecular biology applications Target protein is not exposed to host genome; toxic intermediate states allowed. K D WT = 80nM.

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YSD: Engineering Molecular Interactions

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  1. YSD: Engineering Molecular Interactions • Target protein is on the surface – biophysical characterization of binding by flow cytometry • High-throughput & versatile molecular biology applications • Target protein is not exposed to host genome; toxic intermediate states allowed

  2. KDWT = 80nM Epitope-Normalized MFI KD-6A = 2mM [dsAni WT] in nM YSD of I-Ani1: Analysis of Binding Affinity & Cleavage TGAGGAGGTTTCTCTGTAA TGAGAAGGTTTCTCTGTAA dsOligo ssOligo Cleaved dsOligo

  3. Strategy for Generating a gc-specific variant of I-Ani1 -10-9-8-7-6-5-4-3-2-1 1 2 3 4 5 6 7 8 9 WT = TGAGGAGGTTTCTCTGTAA gc = AAGGAAGGCTTCTCTGTAA • Jim Havranek: Computational redesign of STS1 & 2 • Strategy to generate a YSD library of Jim’s designs • Screen for variants that bind the gc (“SCID”) sequence • Specificity profiling and cleavage analysis • Optimization (epPCR) &/or iteration

  4. 160 designs

  5. Strategy for Generating a Large YSD Library of Designed HEs WT = TGAGGAGGTTTCTCTGTAA gc = AAGGAAGGCTTCTCTGTAA GGATGGAGCCTTTRHTATCAGGAAGCAGGGCAAGARATTGCAGTATGATTTATACATTGAGCTGAGCA = STS1: 80 oligos = 350 variants TATTGGCATCGTAGAATTCAGGAAGAGAAACGAGATTGAAATGGTTGMATTGARSATCAVSGATAAGAATCAT = STS2: 75 oligos = 250 variants Lib Size: 80,000 (STS1 x STS2)

  6. Sorted: Lib 1.0.B1A1 Sorted: Lib 1.0.B1A2 Sorting STS1/2 Library: 1) Expression; 2) Binding; 3) Specificity STS1 PCR STS2 PCR + Galactose HA-APC Myc-FITC Sorted for JH160 STS1+STS2 lib 1.0.B1

  7. JH160 Lib1.0.B1A2 (WT background) JH160 Lib2.0.B1A2 (Y2 background) F13Y

  8. dsOligo ssOligo Cleaved Analysis of Specificity of Individual Clones WT gc

  9. WT Oligo-AF647 SCID Oligo-PE epPCR + combo library Anti-Myc-FITC Employing Counter-selective Sorting to Isolate Specific Binders Pool of high-affinity (non-selective) variants

  10. Round 1 Round 2 Round 3 WT background SCID oligo SCID oligo SCID oligo SCID oligo SCID oligo SCID oligo Myc WT oligo Myc WT oligo Myc WT oligo Y2+L156R background SCID oligo SCID oligo SCID oligo SCID oligo SCID oligo SCID oligo Myc WT oligo Myc WT oligo Myc WT oligo Because I always remake my libraries on the WT enzyme background, there’s always a bit of contamination with yeast that have recombined back in the WT sequence that was cut out…this actually serves as a good internal control for what a specific enzyme should look like

  11. Y2+L156R background

  12. Does Direct Readout Mediate all of the Binding Specificity of NTD? What’s different between the NTD and CTD of I-Ani1?

  13. Does Direct Readout Mediate all of the Binding Specificity of NTD? What’s different between the NTD and CTD of I-Ani1?

  14. What Control’s the Specificity at the NTD of I-Ani1? D73

  15. -10 -9 -8 -7 -6 -5 -4 -3 WT D73G +3 +4 +5 +6 +7 +8 +9 WT D73G D73G: Reduced Specificity of NTD

  16. N20 N20 N20 N4 polar polar Rotamers DG = 0 Rigid DG > 0 +/- - only hydrophobic CH3 or CH Protein-Protein vs. Protein-DNA Interactions • DNA surfaces exhibit much less structural and electrochemical diversity / A2 • DDG (specific vs. non-specific) is lower in DNA-protein interactions • For protein-DNA interactions: specificity is not a simple function of affinity…

  17. Barcoding Yeast for Parallel Analysis of Variant HEs Alexa-350 dsOligo-PE Alexa-647 Myc-FITC Epitope-Normalized MFI [dsOligo]

  18. Overview • DNA-protein interactions • Yeast surface display of I-Ani1 • Hypothesis-driven studies on specificity • Engineering novel DNA-protein interactions

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