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% Cloned Target: 905 Analytical Expressed & Soluble: 215 Purification: 224 Aggregation: 33 Crystal Trial: 63 NMR Screening: 51 PDB (Xray, NMR): 6,7

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 - 1 H/ 2 H exchange mass spectrometry

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

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

10. Digest LC-MS H2OH2O D2OD2O Peptide Mass shift based on D 2 O 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

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

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

13. 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

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

15. Target HTP Construct Design (DisMeta Prediction) Multiple Alternative Constructs Protein Production NMR Screening Xtal 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 tag 57 - 199 + C tag

18. ER541 1-10337-162 1-146 Full length 1-170 Full length Salvage using DisMeta predictions Failed Competition BjR38

19. ER553 SaR32VpR68 LkR15 Salvage using DX-MS results 1-2071-137 1-1361-107 59-199 10 – 90 Micro-probe

20. 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: 216 Purification: 132 Aggregation: 74 Crystal Trial: 49 NMR Screening (Good, Promising): 55(11, 12) PDB (Xray, NMR): 7,4

21. 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: 216 Purification: 132 Aggregation: 74 Crystal Trial: 49 NMR Screening (Good, Promising): 55(11, 12) PDB (Xray, NMR): 7,4

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

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. Twice the time using 1/20 th 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

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 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 Gaetano T. Montelione