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Yuanpeng (Janet) Huang Northeast Structural Genomics Consortium(NESG) Rutgers University

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).

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Yuanpeng (Janet) Huang Northeast Structural Genomics Consortium(NESG) Rutgers University

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  1. 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

  2. PSI-1 NESG-RUTGERS HUMAN PROTEIN PRODUCTION in E. Coli (2000.7.1-2005.7.1) Total PDB/Cloned Targets = 1.4%

  3. 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

  4. 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

  5. SG of extracellular & membrane-bound HCPIN targets -- Chiang, Rossi, Gurla, Montelione, & Anderson, in preparation.

  6. 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

  7. 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

  8. www-nmr.cabm.rutgers.edu/bioinformatics/disorder

  9. Disorder Prediction MetaServer SyR11 Secondary Structure Prediction 1-155 full length Residue 50 Summary of Disorder predictions 50-155truncated Disorder Prediction Server Results difference

  10. Non-deuterated 100 100 575.76 576.24 Relative Abundance Relative Abundance 576.76 577.26 0 0 577.78 574.5 575.0 575.5 576.0 576.5 577.0 577.5 578.0 578.5 579.0 579.5 580.0 m/z Deuterated 576.30 576.78 577.28 575.82 577.78 578.24 578.74 579.24 574.5 575.0 575.5 576.0 576.5 577.0 577.5 578.0 578.5 579.0 579.5 m/z H/D Exchange MS: Concept H. Zheng Digest LC-MS Centroid of peak = 576.13 H2O Quench (pH ~2.5, -80°C) Digest LC-MS Centroid of peak = 576.7 (on ice) D2O Peptide Mass shift based on D2O exposure duration Sharma, et al Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry, Proteins 2009 (in press)

  11. WR33 DXMS Analysis < = 25% > 25% > 50% > 60% > 70% > 80% N N C C C C. elegans, WR33, NESG, R. Tejero, J. Aramini Mouse Homologue, Kobayashi N., et.al Human Homologue, HR387, NESG, J. Aramini

  12. Target HTP Construct Design (DisMeta Prediction) Multiple Alternative Constructs Protein Production Xtal Screening NMR Screening PROTEIN PRODUCTION PROTOCOL Construct Optimization By DXMS/DisMeta

  13. HTP Human Protein Construct Design • 1. Select target domains • PDB hit regions (<80% seq. id) • PFAM domains pdb hits (>80%) pdb hits (<80%) PFAM 2. Find multiple target regions (DisMeta prediction) 3. Propose alternative constructs Total number of constructs (domain) ≈ # of target regions × # of alternative constructs

  14. Total number of constructs (domain) ≈ # of target regions × # of alternative constructs 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 domain • Adjust 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 target domain target region disordered disordered ✗ helix

  15. Target HTP Construct Design (DisMeta Prediction) Multiple Alternative Constructs Protein Production Xtal Screening NMR Screening PROTEIN PRODUCTION PROTOCOL Construct Optimization By DXMS/DisMeta

  16. NMR SALVAGE PROTOCOL

  17. ER553 61 - 199 + C tag 63 - 199 + C tag 59 - 199 + C tag 65 - 199 + C tag57 - 199 + C tag

  18. Salvage using DisMeta predictions BjR38 ER541 1-146 Full length 1-170 Full length Failed Competition 1-103 37-162

  19. ER553 LkR15 SaR32 VpR68 Salvage using DX-MS results 1-207 1-137 1-136 1-107 10 – 90 Micro-probe 59-199

  20. PROGRESS ON PSI-2 RUTGERS HUMAN PROTEIN PRODUCTION (2005.7.1-PRESENT) Total PDB/Cloned Targets (Constructs) = 3%(1.5%)

  21. PROGRESS ON PSI-2 RUTGERS HUMAN PROTEIN PRODUCTION (2005.7.1-PRESENT) Total PDB/Cloned Targets (Constructs) = 3%(1.5%)

  22. SPINE-based Tools HTP robotic NMR micro cryoprobe screening using microgram quantities of protein GVT Swapna Setup & Run Protein samples in Microtubes assessed and scored prior to loading the automatic sample changer Data Archival 1D proton spectrum with water suppression assess Signal-to-noise, foldedness of protein Virtual 96-well Bruker Icon-NMR 2D NH-HSQC spectrum to evaluate the feasibility for structure determination B600 with samples loaded for data collection Archieval of the raw data along with the spectral images, quality scores and stability into SPINE database.

  23. HTP Buffer Optimization Proteins with Good HSQC Precipitation (button testing) Robotic screening using 12 Buffers varying pH, NaCl, Arginine, Acetonitrile, Zn, Ca, Detergent among others. Clear Cloudy precipitated

  24. 3D Structure Determination using microgram quantities of protein • 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 Twice the time using 1/20th of the sample Aramini et al (2007) Microgram-scale protein structure determination by NMR. Nature Methods. 4:491-3

  25. 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

  26. ACKNOWLEDGEMENTS Gaetano T. Montelione Salvage by DXMS Will Buchwald Asli Ertekin Seema Sharma Haiyan Zheng Peter Lobel Bioinformatics John Everett Jessica Locke Binchen Mao Sai Tong Protein Production Thomas Acton Li-Chung Ma Ritu Shastry GVT Swapna Rong Xiao Li Zhao

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