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Clemson CI Symposium - Feb 13, 2013

Establishing a NMR Structural Biology Center at Claflin University. Raj Bhaskaran Department of Chemistry Claflin University, Orangeburg, SC-29115. Clemson CI Symposium - Feb 13, 2013. NMR in CI Symposium. Aim of this talk is two fold:

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Clemson CI Symposium - Feb 13, 2013

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  1. Establishing a NMR Structural Biology Center at Claflin University. Raj Bhaskaran Department of Chemistry Claflin University, Orangeburg, SC-29115 Clemson CI Symposium - Feb 13, 2013

  2. NMR in CI Symposium Aim of this talk is two fold: To attract the Cyber Specialists to join hands with Claflin NMR team to mutually collaborate in the areas that require enormous computing and CI efforts; To attract Life Science researchers to interact with the NMR team to explore their problems structurally to provide a functional meaning; . . . to initiate GEAR CI collaborations. Clemson CI Symposium - Feb 13, 2013

  3. Claflin’s NMR Route to Structural Biology and Drug Discovery Research Claflin has the high field 700 MHz NMR Spectrometer- BioNMR/ Metabolomics NSF Major Research Instrumentation grant is applied for a Cryoprobe and Sample Changer Solution Structure Determination and Protein Dynamics- Functional Interpretation based on structural investigations Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions Protein Structures Derived Functional Interpretation on t. Thermophilusproteome, from Riken, Japanand Filariasis Nematode, BrugiaMalayiproteome, (NIH, UIC) Rapid Protein Structure Determination for Structural Genomics Consortium: Structural Bioinformatics (North East Structural Genomics Consortium; Seattle Structural Genomics Center for Infectious Diseases) Computer Aided Drug Discovery: Virtual screening, Data Mining, QSAR Structure Based Drug Design: SAR by NMR, NMR Screening Clemson CI Symposium - Feb 13, 2013

  4. Structure Based Drug Design • Suitable protein target • Structure of the target protein • Implementation of an easy and reliable HTS assay • Identification of a lead compound • Computer assisted methods for estimating the affinity of new compounds • Access to a synthetic route to produce designed compounds. • Knowledge on shape of the pocket to design the shape of the drug. • The use of X-ray / NMR structures of Protein-Drug Complexes to design better-fitting, and hence more potent inhibitors New Compound Biological Activity Drug-Protein Complex Suggests New Interaction Clemson CI Symposium - Feb 13, 2013

  5. Ligands in the Drug Design Process Identified & Validated Target (genomics, transgenics etc) Free state Structure Protein Production Ligand-Protein structure low affinity, high Kd Assays /HTS morechemistry compound libraries natural products directed libraries more structure high affinity, low Kd Optimised Ligand Hits Chemical tractability Toxicity DrugMetabolism / PharmacoKinetics Clemson CI Symposium - Feb 13, 2013

  6. Extensive computations involved in Protein NMR Study Isotopically15N, 13C, Labeled NMR sample Acquisition of 3D heteronuclear (H, 15N, 13C) NMR experiments Backbone (HN, 15N, 13Ca, Ha ) and Side Chain (13Cb, Hb, 13Cg, Hg , …..) resonance assignment Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 A0 radius) Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol Structural Refinement using additional experimental restraints Protein Dynamics from 15N, 13CRelaxation experiments Clemson CI Symposium - Feb 13, 2013

  7. NMR EXPTS SOFTWARE Backbone NHSQC; CHSQC; TROSYHNCOCA; HNCA HACACONH; HNNCAHA CBCACONH; HNCACB HNCO; HCACOCANH Side chain HBCBCGCD(CE)HD(HE) CCH TOCSY HCCCONH; CCONH HCCH COSY; HCCH TOCSY HCCH TOCSY (aro) NOE N15 EDITED 3D NOESY C13 EDITED 3D NOESY NoesyChsqc (aro) VNMR TOPSPIN NMRPIPE SPARKY AUTO ASSIGN PYMOL / MOLMOL SHIFTX CSI TALOS-PLUS CYANA XPLOR-NIH HADDOCK MODELFREE PROCHECK/PSVS HUMAN ( E219A) MMP-12: 1H-15N HSQC SPECTRUM / NOE CONNECTIVITIES / CSI Bhaskaran & VanDoren JBNMR (2006) Clemson CI Symposium - Feb 13, 2013

  8. First NMR structure of the MMP-12 catalytic domain sans inhibitor RCSB PDB code 2POJ 2803 NOEs Used BB RMSD 0.32 A SC RMSD 0.68 A Ramachandran plot statistics: 77.3% in most favored regions 21.5% in allowed regions 0.0% in disallowed regions active site helix B & -strands I, III are closer in free state NMR – Green; Inhibitors pull B away from the far side X-RAY – Red; 29 unique NOEs ( dotted lines) account for the conformational adjustment Bhaskaran et al. J Mol Biol (2007) Clemson CI Symposium - Feb 13, 2013

  9. Protein Dynamics - rigidity of catalytic domain affect activity upon elastin & 1-AT. L2/3 L3/4 F157 β3 β5 D158 β1 β4 β2 S225 L229 ps-ns MMP-12 MMP-3 Limited Conformational Flexibility High Rigidity MMP-12 is more rigid than MMP-3. Apparent Trade-off of Activity and Rigidity in MMP12 for Stability and Flexibility in MMP3 Liang, Bhaskaran et al Biophysical Jl. (2010) Clemson CI Symposium - Feb 13, 2013

  10. Family of NMR structures of STT3P C-ter Domain -the Catalytic Subunit of Oligo Sachcharyl Transferase RCSB PDB code “2lgz” “WWDYG”motif Ost1p binding site / CW 110 deg Catalyticcenter “DK”motif Membrane embedding site Ave BB rmsd to mean: 0.230 nm Ave heavy atom rmsd to mean : 0.307 nm It is so far the biggest monomeric helical integral membrane protein structure by NMR Huang, Bhaskaran, et al Jl.Biol.Chem.(2012) Clemson CI Symposium - Feb 13, 2013

  11. Structural Gallery - Proteins COBROTOXIN (1COD) CARDIOTOXIN III (2CRS) CARDIOTOXIN II (1CRE) TMH0916 (1NR3) GIP / L-GLUT COMPLEX (2L4T) GIP (2L4S) MMP-12 (2POJ) STT3P (2LGZ) Clemson CI Symposium - Feb 13, 2013

  12. PERTURBATION OF MMP12 BY Triple Helical Peptide AND THE MAPPING OF THEM ON THE STRUCTURE OF MMP12. Binding Isotherm Clemson CI Symposium - Feb 13, 2013 Bhaskaran et al. J Biol Chem.(2008)

  13. Protein:Ligand Interface Mapping by NMR The NMR paramagnetic surface protection assay correctly predicts the sites of interaction as confirmed by the loss of function mutation studies Palmier, Bhaskaran et al Jl Biol Chem. (2010) Clemson CI Symposium - Feb 13, 2013

  14. EVOLUTIONARY TRACE ANALYSIS Evolutionary Trace Method uses a sequence similarity tree of a family of homologous proteins to highlight residues that are statistically under evolutionary pressure and therefore possess certain functional or structural importance for the family Clemson CI Symposium - Feb 13, 2013

  15. BINDSIght Method - maps of binding sites and distinctive sequence to suggest residues tuning specificity Palmier, Bhaskaran et al. J. Biol. Chem. (2010) Clemson CI Symposium - Feb 13, 2013

  16. CI: Scope and Problems for Interaction Pulse program conversion - Varian and Bruker Consolidation of SW packages on NMR structure determination, dynamics and ligand interactions Develop multiuser remote instrumentation to convert into a state level facility A Virtual Web Based File system for managing NMR Data Rapid structure determination package for the structural genomics proteins Clemson CI Symposium - Feb 13, 2013

  17. BioNMR – External Collaborations Dr. Steven VanDoren, University of Missouri, Columbia, MO : Structural Interactions of Tumor Viral Proteins, E7 and E2F Dr.K.Ramaswamy, UIC, Rockford, IL : Structural Studies of Filarial Proteins Bm-HSP 12.6 Dr.M.Gnanasekar, UIC, Rockford, IL : Structural Studies of Filarial Proteins Bm-TCTP Dr. Peter Myler, Seattle Biomed, Seattle, WA : Structural Genomics of Proteins from Infectious Diseases Dr. Krishna Sharma, University of Missouri, Columbia, MO : Structures of a-Crystallin & Chaperonins of Eye Disease, Cataract Dr. Raghu Kannan, University of Missouri, Columbia, MO : NMR of Gold Nanoparticle Conjugated Bombesin Dr. T. K. S. Kumar, University of Arkansas, Fayetteville, AR : Structural Studies of Fibroblast Growth factor, FGF2 Dr. K. Gunasekaran, University of Madras, Chennai, India : Structural Studies of Filarial Proteins Bm-API Dr. D. Velmurugan, University of Madras, Chennai, India : Structural Genomics of t. thermophilusProteins Dr. P. Karthe, University of Madras, Chennai, India : NMR structure of Serine glutamate repeat A, a surface adhesin Clemson CI Symposium - Feb 13, 2013

  18. GEAR- CI: SC-Collaborations Dr. John Dawson, University of South Carolina : Identification of Dehaloperoxidase-Substrate Binding Sites Dr. Caryn Outten, University of South Carolina : Structural Studies of iron sensing proteins, Fra2 and Grx Dr.HomayounValafar, University of South Carolina : NMR Residual Dipolar Coupling analyses Dr. Nick Grossoheme, Winthrop University: Dr. Heather Evans Anderson, Winthrop University: NMR structure of ForkHead Transcription Factor Protein, FOXO1 Dr. Sondra Berger, University of South Carolina : Dr. Angela Peters, Claflin University : Thymidalate Synthase Mutant (R163K)-Ligand Interactions Dr. Michael Sehorn, Clemson University : Dr. Erin Eaton, Francis Marion University : Dr. Karen Buchmuller, Furman University : Dr. Marcello Forconi, College of Charleston : Dr.EsmaeilJabbari, University of South Carolina : (CRP) NMR Structural Characterization of vasculogenic peptides Dr. Scott Argraves, University of South Carolina : (CRP) NMR Studies of Fibulins, the extracellular matrix proteins Clemson CI Symposium - Feb 13, 2013

  19. TRUSTWORTHINESS RESPECT RESPONSIBILITY FAIRNESS CITIZENSHIP CARING ACKNOWLEDGEMENT Mr.RaghavNagampalli (Fulbright Scholar) Dr. Angela Peters (Claflin) Dr.Verlie Tisdale (Claflin) School of Natural Sciences and Mathematics, Claflin SC INBRE NASA EPSCoR Space Grant. Clemson CI Symposium - Feb 13, 2013

  20. THANK YOU ALL Clemson CI Symposium - Feb 13, 2013

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