YSD: Engineering Molecular Interactions Target protein is on the surface – biophysical...
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Transcript of YSD: Engineering Molecular Interactions Target protein is on the surface – biophysical...
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