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

YSD of I-Ani1: Analysis of Binding Affinity & Cleavage

dsOligo

ssOligo

Cleaved dsOligo

TGAGGAGGTTTCTCTGTAA

TGAGAAGGTTTCTCTGTAA

KDWT = 80nM

[dsAni WT] in nM

Epito

pe-N

orm

aliz

ed M

FI

KD-6A = 2M

1. Jim Havranek: Computational redesign of STS1 & 22. Strategy to generate a YSD library of Jim’s designs 3. Screen for variants that bind the c (“SCID”) sequence4. Specificity profiling and cleavage analysis5. Optimization (epPCR) &/or iteration

Strategy for Generating a c-specific variant of I-Ani1

WT = TGAGGAGGTTTCTCTGTAAc = AAGGAAGGCTTCTCTGTAA

-10-9-8-7-6-5-4-3-2-1 1 2 3 4 5 6 7 8 9

160 designs

GGATGGAGCCTTTRHTATCAGGAAGCAGGGCAAGARATTGCAGTATGATTTATACATTGAGCTGAGCA = STS1: 80 oligos = 350 variants

TATTGGCATCGTAGAATTCAGGAAGAGAAACGAGATTGAAATGGTTGMATTGARSATCAVSGATAAGAATCAT = STS2: 75 oligos = 250 variants

Lib Size: 80,000 (STS1 x STS2)

Strategy for Generating a Large YSD Library of Designed HEs

WT = TGAGGAGGTTTCTCTGTAAc = AAGGAAGGCTTCTCTGTAA

STS1 PCR STS2 PCR

Myc-FITC

HA-

APC

Sorted for JH160 STS1+STS2 lib 1.0.B1

Sorted: Lib 1.0.B1A2

0

500

1000

1500

2000

2500

3000

1 10 100 1000 10000[dsAniSCID] nM

Myc

-Nor

mal

ized

MFI

JH160 Lib1.0.B1A1

JH160 Lib1.0.B1A2

JH160 Lib1.0.B1

+ Galactose

Sorting STS1/2 Library: 1) Expression; 2) Binding; 3) Specificity

Sorted: Lib 1.0.B1A1

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

F13Y

Analysis of Specificity of Individual Clones

WT

c

dsOligo

ssOligo

Cleaved

Employing Counter-selective Sorting to Isolate Specific Binders

Pool of high-affinity (non-selective) variants

WT Oligo-AF647

SCID Oligo-PE

Anti-Myc-FITC epPCR + combo library

Round 1 Round 2 Round 3SC

ID o

ligo

SCID

olig

o

Myc WT oligo

SCID

olig

o

SCID

olig

o

Myc WT oligo

SCID

olig

o

SCID

olig

o

Myc WT oligo

SCID

olig

o

SCID

olig

o

Myc WT oligo

SCID

olig

o

SCID

olig

o

Myc WT oligo

SCID

olig

o

SCID

olig

o

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

WT

back

grou

ndY2

+L15

6R b

ackg

roun

d

Y2+L156R background

Does Direct Readout Mediate all of the Binding Specificity of NTD?

What’s different between the NTD and CTD of I-Ani1?

What’s different between the NTD and CTD of I-Ani1?

Does Direct Readout Mediate all of the Binding Specificity of NTD?

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

D73

D73G: Reduced Specificity of NTD

WT

WT

D73G

D73G

-10 -9 -8 -7 -6 -5 -4 -3

+4 +3 +6 +5 +7 +9 +8

Protein-Protein vs. Protein-DNA Interactions

N20 N20 N20 N4

+/- hydrophobic

polar - only

CH3 or CH

polar Rotamers G = 0

Rigid G > 0

• DNA surfaces exhibit much less structural and electrochemical diversity / A2

• G (specific vs. non-specific) is lower in DNA-protein interactions• For protein-DNA interactions: specificity is not a simple function of affinity…

Barcoding Yeast for Parallel Analysis of Variant HEs

Alexa-647

Alex

a-35

0

[dsOligo]

Epito

pe-N

orm

aliz

ed M

FI

Myc-FITC

dsO

ligo-

PE

Overview

1. DNA-protein interactions

2. Yeast surface display of I-Ani1

3. Hypothesis-driven studies on specificity

4. Engineering novel DNA-protein interactions