HTP Construct Optimization using Bioinformatics Coupled with Amide Hydrogen Deuterium Exchange (DXMS) and HTP NMR screening
Yuanpeng (Janet) Huang
Northeast Structural Genomics Consortium(NESG)
Rutgers University
PSI-1 NESG-RUTGERS HUMAN PROTEIN PRODUCTION in E. Coli (2000.7.1-2005.7.1)
Total PDB/Cloned Targets = 1.4%
Cloned Target: 905 Analytical Expressed & Soluble: 215Purification: 224Aggregation: 33Crystal Trial: 63NMR Screening: 51PDB (Xray, NMR): 6,7
PSI-2 BIOMEDICAL THEME – Human Cancer Protein Interaction Network (HCPIN)
Huang, et al (2008) Targeting the human cancer pathway protein interaction network by structural genomics Molecular & Cellular Proteomics 7: 2048-2060
EFFORTS TO IMPROVE PSI-2 HUMAN PROTEIN PRODUCTIVITY
Target Selection Select proteins validated by SwissProt Exclude proteins annotated or predicted to be
secreted or TM Gene synthesis and RT-PCR Construct Design and Optimization
Identify disordered regions by DisMeta prediction DXMS analysis
SG of extracellular & membrane-bound HCPIN targets
-- Chiang, Rossi, Gurla, Montelione, & Anderson, in preparation.
EFFORTS TO IMPROVE PSI-2 HUMAN PROTEIN PRODUCTIVITY
Target Selection Select proteins validated by SwissProt Exclude proteins annotated or predicted to be
secreted or TM Gene synthesis and RT-PCR Construct Optimization
Identify disordered regions by DisMeta prediction DXMS analysis
SOME PARTIALLY DISORDERED PROTEIN STRUCTURES SOLVED BY NESG
Interfere with Structural Determination Efforts
Identify disordered regions
DisMeta - Disorder Prediction MetaServer
DXMS - 1H/2H exchange mass spectrometry
www-nmr.cabm.rutgers.edu/bioinformatics/disorder
Secondary Structure Prediction
Disorder Prediction Server Results
Summary of Disorder predictions
SyR11
Residue 50
Disorder Prediction MetaServer
1-155 full length
50-155truncated
difference
Digest
Digest
LC-MS
LC-MS
H2O
D2O
m/z574.5 575.0 575.5 576.0 576.5 577.0 577.5 578.0 578.5 579.0 579.5 580.0
575.76
576.24
576.76
577.26577.78
0
100
Rela
tive A
bund
ance
Non-deuterated
m/z574.5 575.0 575.5 576.0 576.5 577.0 577.5 578.0 578.5 579.0 579.5
576.30
576.78
577.28
575.82
577.78
578.24578.74579.240
100
Rela
tive A
bund
ance
Deuterated
Peptide Mass shift based on D2O exposure duration
H/D Exchange MS: Concept
Quench
(pH ~2.5, -80°C)
(on ice)Centroid of peak = 576.7
Centroid of peak = 576.13
Sharma, et al Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry, Proteins 2009 (in press)
H. Zheng
WR33 DXMS Analysis
> 50%
< = 25%
> 25%
> 60%
> 70%
> 80%
Mouse Homologue, Kobayashi N., et.al
C. elegans, WR33, NESG,
R. Tejero, J. Aramini
CNN
C
Human Homologue, HR387, NESG,
J. Aramini
C
Target
HTP Construct Design (DisMeta Prediction)
Multiple Alternative Constructs
ProteinProduction
NMR Screening
Xtal Screening
PROTEIN PRODUCTION PROTOCOL
Construct OptimizationBy DXMS/DisMeta
HTP Human Protein Construct Design
pdb hits (>80%)
2. Find multiple target regions(DisMeta prediction)
3. Propose alternative constructs
1. Select target domains•PDB hit regions (<80% seq. id) •PFAM domains
pdb hits (<80%) PFAM
Total number of constructs (domain) ≈ # of target regions × # of alternative constructs
target region
disordered
Propose Alternative Constructs
Identify multiple target regions (TRs) for each target domain All TRs with length > 50aa and cover at least 80% of
the target domainAdjust TR for disorder and helix/strand regions
For each TR (S,E)Propose 1-4 alternative constructs (S-5, S)x(E, E+5)Remove the ones intersect with helix/strand Adjust N/C ends (-2:2)
target domain
disordered
target domain
helix✗
Total number of constructs (domain) ≈ # of target regions × # of alternative constructs
Target
HTP Construct Design (DisMeta Prediction)
Multiple Alternative Constructs
ProteinProduction
NMR Screening
Xtal Screening
PROTEIN PRODUCTION PROTOCOL
Construct OptimizationBy DXMS/DisMeta
NMR SALVAGE PROTOCOL
ER553
61 - 199 + C tag 63 - 199 + C tag 59 - 199 + C tag 65 - 199 + C tag57 - 199 + C tag
ER541
1-103 37-162
1-146Full length
1-170Full length
Salvage using DisMeta predictions
Failed Competition
BjR38
ER553
SaR32 VpR68
LkR15
Salvage using DX-MS results
1-207 1-137
1-136 1-10759-199 10 – 90
Micro-probe
PROGRESS ON PSI-2 RUTGERS HUMAN PROTEIN PRODUCTION (2005.7.1-PRESENT)
Total PDB/Cloned Targets (Constructs) = 3%(1.5%)
Cloned Target (Construct): 367 (734)Analytical Expressed & Soluble: 216Purification: 132Aggregation: 74Crystal Trial: 49NMR Screening (Good, Promising): 55(11, 12)PDB (Xray, NMR): 7,4
PROGRESS ON PSI-2 RUTGERS HUMAN PROTEIN PRODUCTION (2005.7.1-PRESENT)
Total PDB/Cloned Targets (Constructs) = 3%(1.5%)
Cloned Target (Construct): 367 (734)Analytical Expressed & Soluble: 216Purification: 132Aggregation: 74Crystal Trial: 49NMR Screening (Good, Promising): 55(11, 12)PDB (Xray, NMR): 7,4
HTP robotic NMR micro cryoprobe screening using microgram quantities of protein
Protein samples inMicrotubes assessed and scored prior to loading theautomatic sample changer
B600 with samples loadedfor data collection
1D proton spectrum with water suppressionassess Signal-to-noise, foldedness of protein
2D NH-HSQC spectrum to evaluatethe feasibility for structure determination
Archieval of the raw data along with thespectral images, quality scores and stabilityinto SPINE database.
Setup
&
Run
Data
Archival
Virtual 96-well
SPINE-based Tools
Bruker Icon-NMR
GVT Swapna
HTP Buffer Optimization
MJ001 MJ002 MJ003 MJ004 MJ005 MJ006 MJ007 MJ008 MJ009 MJ010 MJ011 MJ012
A CcR27.008 CcR27.009 CcR27.010 CcR27.011 CcR27.012 CcR27.013 CcR27.014 CcR27.015 CcR27.016 CcR27.017 CcR27.018 CcR27.019
B StR82.012 StR82.013 StR82.014 StR82.015 StR82.016 StR82.017 StR82.018 StR82.019 StR82.020 StR82.021 StR82.022 StR82.023
C SaR31.005 SaR31.006 SaR31.007 SaR31.008 SaR31.009 SaR31.010 SaR31.011 SaR31.012 SaR31.013 SaR31.014 SaR31.015 SaR31.016
D VpR162.008 VpR162.009 VpR162.010 VpR162.011 VpR162.012 VpR162.013 VpR162.014 VpR162.015 VpR162.016 VpR162.017 VpR162.018 VpR162.019
E MaR182.008 MaR182.009 MaR182.010 MaR182.011 MaR182.012 MaR182.013 MaR182.014 MaR182.015 MaR182.016 MaR182.017 MaR182.018 MaR182.019
F SvR198.008 SvR198.009 SvR198.010 SvR198.011 SvR198.012 SvR198.013 SvR198.014 SvR198.015 SvR198.016 SvR198.017 SvR198.018 SvR198.019G IR12.004 IR12.005 IR12.006 IR12.007 IR12.008 IR12.009 IR12.010 IR12.011 IR12.012 IR12.013 IR12.014 IR12.015
MJ001 MJ002 MJ003 MJ004 MJ005 MJ006 MJ007 MJ008 MJ009 MJ010 MJ011 MJ012
A p p p
B p p P P P P P P p p P
C p p P p P p p p p
D p p P P P P P p p P
E p p p P p p p
F p p P P p P p p P p
G p p P P P p P p p
1D Proton Experiment Good = Perform 2D HSQC Bad = Stop WorkMJ001 MJ002 MJ003 MJ004 MJ005 MJ006 MJ007 MJ008 MJ009 MJ010 MJ011 MJ012
A p p p
B p p P P P P P P p p P
C p p P p P p p p p
D p p P P P P P p p P
E p p p P p p p
F p p P P p P p p P p
G p p P P P p P p p
Proteins with Good HSQCPrecipitation (button testing)
Robotic screening using12 Buffers varying pH, NaCl, Arginine, Acetonitrile, Zn, Ca, Detergent among others.
Clear Cloudy precipitated
Twice the time using 1/20th of the sample
1 mm micro probe and 1.7 mm micro cryoprobe
It is now routinely used in NESG HTP structure production pipeline
• low yield eukaryotic proteins
3D Structure Determination using microgram quantities of protein
Aramini et al (2007) Microgram-scale protein structure determination by NMR. Nature Methods. 4:491-3
SUMMARY Current protocol of construct optimization is focused
on identification of disorder regions DisMeta
Very fast and no-cost HTP construct design protocol is developed together with DisMeta
DXMS More reliable, experimental evidence on disordered regions Useful for identification of disordered regions when the prediction
is not satisfactory Automated analysis of DXMS data is under development
Human protein production in E. Coli is improved NMR micro cryoprobe Efficient and cost-effective HTP robotic NMR screening NMR Structure determination become feasible for human
proteins with low expression and low solubility
ACKNOWLEDGEMENTS
Salvage by DXMSWill BuchwaldAsli ErtekinSeema SharmaHaiyan ZhengPeter Lobel
BioinformaticsJohn EverettJessica LockeBinchen MaoSai Tong
Protein Production Thomas ActonLi-Chung MaRitu ShastryGVT SwapnaRong XiaoLi Zhao
Gaetano T. Montelione
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