Clemson CI Symposium - Feb 13, 2013
Establishing a NMR Structural Biology Establishing a NMR Structural Biology Center at Claflin University.Center at Claflin University.
Raj BhaskaranRaj BhaskaranDepartment of Chemistry Department of Chemistry
Claflin University, Orangeburg, SC-29115Claflin University, Orangeburg, SC-29115
NMR in CI SymposiumNMR in CI Symposium
Clemson CI Symposium - Feb 13, 2013
Aim of this talk is two fold:Aim of this talk is two fold:
To attract the Cyber Specialists to join hands with Claflin To attract the Cyber Specialists to join hands with Claflin NMR team to mutually collaborate in the areas that require NMR team to mutually collaborate in the areas that require enormous computing and CI efforts;enormous computing and CI efforts;
To attract Life Science researchers to interact with the To attract Life Science researchers to interact with the NMR team to explore their problems structurally to NMR team to explore their problems structurally to provide a functional meaning;provide a functional meaning;
. . . to initiate GEAR CI . . . to initiate GEAR CI collaborationscollaborations..
Claflin’s NMR Route to Claflin’s NMR Route to Structural Biology and Drug Discovery ResearchStructural Biology and Drug Discovery Research
Clemson CI Symposium - Feb 13, 2013
ClaflinClaflin has the high field 700 MHz NMR Spectrometer- BioNMR/ has the high field 700 MHz NMR Spectrometer- BioNMR/ MetabolomicsMetabolomics
NSF Major Research Instrumentation grant is applied for a Cryoprobe and NSF Major Research Instrumentation grant is applied for a Cryoprobe and Sample Changer Sample Changer
Solution Structure Determination and Protein Dynamics- Functional Solution Structure Determination and Protein Dynamics- Functional Interpretation based on structural investigationsInterpretation based on structural investigations
Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions
Protein Structures Derived Functional Interpretation on Protein Structures Derived Functional Interpretation on t. Thermophilus t. Thermophilus proteome, from Riken, Japanproteome, from Riken, Japan and and Filariasis Nematode, Filariasis Nematode, Brugia Malayi Brugia Malayi proteome, (NIH, UIC)proteome, (NIH, UIC)
Rapid Protein Structure Determination for Structural Genomics Consortium: Rapid Protein Structure Determination for Structural Genomics Consortium: Structural Bioinformatics (North East Structural Genomics Consortium; Structural Bioinformatics (North East Structural Genomics Consortium; Seattle Structural Genomics Center for Infectious Diseases)Seattle Structural Genomics Center for Infectious Diseases)
Computer Aided Drug Discovery: Virtual screening, Data Mining, QSARComputer Aided Drug Discovery: Virtual screening, Data Mining, QSAR
Structure Based Drug Design: SAR by NMR, NMR ScreeningStructure Based Drug Design: SAR by NMR, NMR Screening
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
Clemson CI Symposium - Feb 13, 2013
New Compound
Suggests New Interaction
Drug-Protein Complex
Biological Activity
Ligands in the Drug Design Process
Clemson CI Symposium - Feb 13, 2013
compound librariesnatural productsdirected libraries
Identified & Validated Target
Protein Production
Assays /HTS
Hits
Chemical tractabilityToxicityDrugMetabolism /
PharmacoKinetics
(genomics, transgenics etc)
more structure
Structure
Ligand-Protein structure
Optimised Ligand
more chemistry
low affinity,high Kd
high affinity,low Kd
Free state
Extensive computations involved in Protein NMR StudyExtensive computations involved in Protein NMR Study
Clemson CI Symposium - Feb 13, 2013
Isotopically Isotopically 1515N, N, 1313C, Labeled NMR sampleC, Labeled NMR sample
Acquisition of 3D heteronuclear (H, Acquisition of 3D heteronuclear (H, 1515N, N, 1313C) NMR experimentsC) NMR experiments
Backbone (HN, Backbone (HN, 1515N, N, 1313CC, H, H ) and Side Chain ( ) and Side Chain (1313CC, H, H1313CC, H, H , …..) resonance assignment , …..) resonance assignment
Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 AA0 0 radius)radius)
Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol
Structural Refinement using additional experimental restraintsStructural Refinement using additional experimental restraints
Protein Dynamics from Protein Dynamics from 1515N, N, 1313CCRelaxation experimentsRelaxation experiments
HUMAN ( E219A) MMP-12: HUMAN ( E219A) MMP-12: 11H-H-1515N HSQC SPECTRUM / NOE CONNECTIVITIES / CSIN HSQC SPECTRUM / NOE CONNECTIVITIES / CSI
Clemson CI Symposium - Feb 13, 2013
Backbone
NHSQC; CHSQC;
TROSY HNCOCA; 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
SOFTWARESOFTWARE NMR EXPTS NMR EXPTS
Bhaskaran & VanDoren JBNMR (2006)
F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G
105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
dN(i, i+1)
dNN(i, i+1)
dN(i, i+2)
d(i, i+3)
dN(i, i+3)
Secondary Structure
F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G
105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G
105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185
dN(i, i+1)
dNN(i, i+1)
dN(i, i+2)
d(i, i+3)
dN(i, i+3)
Secondary Structure
P G S G I G G D A H F D E D E F W T T H S G G T N L F L T A V H A I G H S L G L G H S S D D K A V M F D T Y K Y V D I N T F R L S A D D I R G I Q S L Y G
190 195 200 205 210 215 220 225 230 235 240 245 250 255 260
dN(i, i+1)
dNN(i, i+1)
dN(i, i+2)
d(i, i+3)
dN(i, i+3)
Secondary Structure
P G S G I G G D A H F D E D E F W T T H S G G T N L F L T A V H A I G H S L G L G H S S D D K A V M F D T Y K Y V D I N T F R L S A D D I R G I Q S L Y G
190 195 200 205 210 215 220 225 230 235 240 245 250 255 260
P G S G I G G D A H F D E D E F W T T H S G G T N L F L T A V H A I G H S L G L G H S S D D K A V M F D T Y K Y V D I N T F R L S A D D I R G I Q S L Y G
190 195 200 205 210 215 220 225 230 235 240 245 250 255 260
dN(i, i+1)
dNN(i, i+1)
dN(i, i+2)
d(i, i+3)
dN(i, i+3)
Secondary Structure
110 130 150 170 190 210 230 250 120 140 160 180 200 220 240 260
0.6
0.4
0.2
0.0
- 0.2
- 0.4
MMP-12 Residue
110 130 150 170 190 210 230 250 120 140 160 180 200 220 240 260
0.6
0.4
0.2
0.0
- 0.2
- 0.4
MMP-12 Residue
First NMR structure of the MMP-12 catalytic domain sans inhibitor
Clemson CI Symposium - Feb 13, 2013 Bhaskaran et al. J Mol Biol (2007)
29 unique NOEs( dotted lines)account for the conformationaladjustment
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;
RCSB PDB code 2POJ2803 NOEs UsedBB RMSD 0.32 ASC RMSD 0.68 A
Ramachandran plot statistics:
77.3% in most favored regions
21.5% in allowed regions
0.0% in disallowed regions
Protein Dynamics - rigidity of catalytic domain affect activity upon elastin & 1-AT.
High Rigidity Limited Conformational Flexibility
Clemson CI Symposium - Feb 13, 2013
MMP-12 MMP-3
ps-ns
110 120130 140 150 160 170 180 190 200 210 220 230 240 250 2600.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Order param eter S2 for MMP-12
54321
S2
Residue Number
β1
β2
β3
β4
β5
S225
L229
D158
F157
L2/3 L3/4
90 1001101201301401501601701801902002102202302402502600.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Order parameter S2 for MMP-3
S2
Residue Number
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)
Family of NMR structures of STT3P C-ter Domain -the Catalytic Subunit of Oligo Sachcharyl Transferase
Ave BB rmsd to mean: 0.230 nmAve heavy atom rmsd to mean : 0.307 nm
Clemson CI Symposium - Feb 13, 2013 Huang, Bhaskaran, et al Jl.Biol.Chem. (2012)
RCSB PDB code “2lgz”
It is so far the biggest monomeric helical integral membrane protein structure by NMR
/
Ost1p binding site
Membrane embedding siteCatalytic center
“WWDYG”motif
“DK”motif
CW 110 deg
Structural Gallery - Proteins
COBROTOXIN (1COD) CARDIOTOXIN III (2CRS)
TMH0916 (1NR3) GIP / L-GLUT COMPLEX (2L4T) GIP (2L4S)
MMP-12 (2POJ) STT3P (2LGZ)
CARDIOTOXIN II (1CRE)
Clemson CI Symposium - Feb 13, 2013
PERTURBATION OF MMP12 BY PERTURBATION OF MMP12 BY Triple Helical PeptideTriple Helical Peptide AND THE MAPPING AND THE MAPPING OF THEM ON THE STRUCTURE OF MMP12.OF THEM ON THE STRUCTURE OF MMP12.
Chemical Shift Perturbation of 1H-15N NMR Correlation Spectrum for Finding Binding Site
15N
1H
Clemson CI Symposium - Feb 13, 2013
Bhaskaran et al. J Biol Chem.(2008)
0.0000 0.0001 0.0002 0.0003
0.00
0.01
0.02
0.03
0.04
0.05K
d = 30 ± 6 M
H(p
pm
)
[1(V)THP] (M)
H112
G221
L147
Y113
T205
K148Y240
L224
T210V243
0.0000 0.0001 0.0002 0.0003
0.00
0.01
0.02
0.03
0.04
0.05K
d = 30 ± 6 M
H(p
pm
)
[1(V)THP] (M)
H112
G221
L147
Y113
T205
K148Y240
L224
T210V243
Binding Isotherm
Clemson CI Symposium - Feb 13, 2013
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)
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
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
CI: Scope and Problems for InteractionCI: Scope and Problems for Interaction
Clemson CI Symposium - Feb 13, 2013
Pulse program conversion - Varian and BrukerPulse program conversion - Varian and Bruker
Consolidation of SW packages on NMR structure Consolidation of SW packages on NMR structure determination, dynamics and ligand interactionsdetermination, dynamics and ligand interactions
Develop multiuser remote instrumentation to convert into Develop multiuser remote instrumentation to convert into a state level facilitya state level facility
A Virtual Web Based File system for managing NMR DataA Virtual Web Based File system for managing NMR Data
Rapid structure determination package for the structural Rapid structure determination package for the structural genomics proteins genomics proteins
BioNMR – External CollaborationsBioNMR – External Collaborations
Clemson CI Symposium - Feb 13, 2013
Dr. Steven VanDoren, University of Missouri, Columbia, MO :Structural Interactions of Tumor Viral Proteins, E7 and
E2FDr. K.Ramaswamy, UIC, Rockford, IL :
Structural Studies of Filarial Proteins Bm-HSP 12.6Dr. M.Gnanasekar, UIC, Rockford, IL :
Structural Studies of Filarial Proteins Bm-TCTPDr. Peter Myler, Seattle Biomed, Seattle, WA :
Structural Genomics of Proteins from Infectious DiseasesDr. Krishna Sharma, University of Missouri, Columbia, MO :
Structures of -Crystallin & Chaperonins of Eye Disease, CataractDr. Raghu Kannan, University of Missouri, Columbia, MO :
NMR of Gold Nanoparticle Conjugated BombesinDr. 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. thermophilus Proteins
Dr. P. Karthe, University of Madras, Chennai, India :NMR structure of Serine glutamate repeat A, a surface
adhesin
GEAR- CI: SC-CollaborationsGEAR- CI: SC-Collaborations
Clemson CI Symposium - Feb 13, 2013
Dr. John Dawson, University of South Carolina :Dr. John Dawson, University of South Carolina : Identification of Dehaloperoxidase-Substrate Binding Sites
Dr. Caryn Outten, University of South Carolina :Dr. Caryn Outten, University of South Carolina :Structural Studies of iron sensing proteins, Fra2 and Grx
Dr. Homayoun Valafar, University of South Carolina :Dr. Homayoun Valafar, University of South Carolina :NMR Residual Dipolar Coupling analyses
Dr. Nick Grossoheme, Winthrop University:Dr. Nick Grossoheme, Winthrop University:Dr. Heather Evans Anderson, Winthrop University:Dr. Heather Evans Anderson, Winthrop University:
NMR structure of ForkHead Transcription Factor Protein, FOXO1Dr. Sondra Berger, University of South Carolina :Dr. Sondra Berger, University of South Carolina :Dr. Angela Peters, Claflin University :Dr. Angela Peters, Claflin University :
Thymidalate Synthase Mutant (R163K)-Ligand Interactions Dr. Michael Sehorn, Clemson University :Dr. Michael Sehorn, Clemson University :
Dr. Erin Eaton, Francis Marion University :Dr. Erin Eaton, Francis Marion University :
Dr. Karen Buchmuller, Furman University :Dr. Karen Buchmuller, Furman University :
Dr. Marcello Forconi, College of Charleston :Dr. Marcello Forconi, College of Charleston :
Dr. Esmaeil Jabbari, University of South Carolina : (CRP)Dr. Esmaeil Jabbari, University of South Carolina : (CRP) NMR Structural Characterization of vasculogenic peptides
Dr. Scott Argraves, University of South Carolina : (CRP)Dr. Scott Argraves, University of South Carolina : (CRP) NMR Studies of Fibulins, the extracellular matrix proteins
ACKNOWLEDGEMENTACKNOWLEDGEMENT
Clemson CI Symposium - Feb 13, 2013
SC INBRE
NASA EPSCoR Space Grant.
Mr. Raghav Nagampalli (Fulbright Scholar)Mr. Raghav Nagampalli (Fulbright Scholar)
Dr. Angela Peters (Claflin) Dr. Angela Peters (Claflin)
Dr. Verlie Tisdale (Claflin)Dr. Verlie Tisdale (Claflin)
School of Natural Sciences and Mathematics, ClaflinSchool of Natural Sciences and Mathematics, Claflin
TRUSTWORTHINESSRESPECT
RESPONSIBILITYFAIRNESS
CITIZENSHIPCARING
THANK YOU ALLTHANK YOU ALL
Clemson CI Symposium - Feb 13, 2013