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:
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
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. Thermophilus
proteome, from Riken, Japan and Filariasis Nematode, Brugia Malayi
proteome, (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
Structure Based Drug Design
•Suitable protein target
New Compound
•Structure of the target protein
Biological Activity
•Implementation of an easy and
reliable HTS assay
•Identification of a lead compound
Suggests
New Interaction
Drug-Protein
Complex
•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
Ligands in the Drug Design Process
Identified & Validated Target
(genomics, transgenics etc)
Protein Production
Assays /HTS
Structure
Ligand-Protein
structure
Free state
low affinity,
high Kd
compound libraries
natural products
directed libraries
Hits
more chemistry
more structure
Optimised Ligand
high affinity,
low Kd
Clemson CI Symposium - Feb 13, 2013
Chemical tractability
Toxicity
DrugMetabolism /
PharmacoKinetics
Extensive computations involved in Protein NMR Study
Isotopically 15N, 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, 13C Relaxation experiments
Clemson CI Symposium - Feb 13, 2013
NMR EXPTS
SOFTWARE
Backbone
VNMR
NHSQC; CHSQC;
TOPSPIN
TROSY HNCOCA; HNCA
HACACONH; HNNCAHA
NMRPIPE
CBCACONH; HNCACB
SPARKY
HNCO; HCACOCANH
AUTO ASSIGN
Side chain
PYMOL / MOLMOL
HBCBCGCD(CE)HD(HE)
SHIFTX
CCH TOCSY
105
F
110
R E M P G G
115
P V W R K H
Y
I
T
120
Y R
I
N N
125
Y T
130
P D M N R
E D
V D Y A
135
I
R K
140
A
F Q
V W S
145
N V
T P
150
L K
F S K
155
I
N T G M A
160
D I
165
L V
V F
170
A R G A H G
175
D F H
A F
180
D G K G G I
daN(i, i+1)
HCCCONH; CCONH
dNN(i, i+1)
HCCH COSY;
daN(i, i+3)
TALOS-PLUS
Secondary Structure
P
G S G
195
I
G G D A H
200
F D
E D E
205
F W T
210
T H S G G
T N
215
L F L T A V
220
H A I G H
225
S
230
L G L G H
S S
235
240
245
D D K A V M F D T Y K Y
V D
I
250
N
T F R L
255
S A D D
260
I
R G
I
Q S
L Y G
daN(i, i+1)
CYANA
dNN(i, i+1)
dab(i, i+3)
XPLOR-NIH
daN(i, i+3)
NOE
N15 EDITED 3D NOESY
Secondary Structure
MMP-12 Residue
0.6
HADDOCK
0.4
0.2
0.0
C13 EDITED 3D NOESY
F G
dab(i, i+3)
daN(i, i+2)
HCCH TOCSY (aro)
A H A
CSI
daN(i, i+2)
190
HCCH TOCSY
185
L
MODELFREE
- 0.2
- 0.4
110
NoesyChsqc (aro)
HUMAN ( E219A) MMP-12:
Clemson CI Symposium - Feb 13, 2013
130
120
150
140
1H-15N
170
160
190
180
210
200
230
220
250
240
260
PROCHECK/PSVS
HSQC SPECTRUM / NOE CONNECTIVITIES / CSI
Bhaskaran & VanDoren JBNMR (2006)
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
& b-strands I, III
are closer in free
state NMR – Green;
Inhibitors pull aB
away from the far
side X-RAY – Red;
Clemson CI Symposium - Feb 13, 2013
29 unique NOEs
( dotted lines)
account for the
conformational
adjustment
Bhaskaran et al. J Mol Biol (2007)
Protein Dynamics - rigidity of catalytic domain affect
activity upon elastin & a1-AT.
O rd er
p a ra m eter S
2
O rd er p a ra met er S
fo r M M P -1 2
b
1 .2
1 .1
b 1
a 
b2
b3
b4
a 
b5
2
fo r M M P-3
1 .2
a
b
b
b
b
a
a
1 .1
a
1 .0
1 .0
0 .9
0 .9
0 .8
0 .8
0 .7
2
0 .7
2
S
0 .5
0 .4
0 .6
S
0 .6
0 .5
ps-ns
0 .4
0 .3
0 .3
0 .2
0 .2
0 .1
0 .0
MMP-12
0 .1
MMP-3
0 .0
90 100 110120 130 140150 160 170 180190 200210 220 230 240250 260
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260
R e sid u e N u m b e r
R e s id u e N u m b e r
High Rigidity
Limited Conformational Flexibility
MMP-12 is more rigid than MMP-3.
L2/3
L3/4
F157
β3
β5
D158
β1
β4
β2
S225
L229
Apparent Trade-off of Activity and Rigidity in MMP12 for Stability and
Flexibility in MMP3
Clemson CI Symposium - Feb 13, 2013
Liang, Bhaskaran et al Biophysical Jl. (2010)
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
Membrane embedding site
/
Catalytic center
“DK”motif
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
Clemson CI Symposium - Feb 13, 2013
Huang, Bhaskaran, et al Jl.Biol.Chem. (2012)
Structural Gallery - Proteins
COBROTOXIN (1COD)
TMH0916 (1NR3)
CARDIOTOXIN III (2CRS)
CARDIOTOXIN II (1CRE)
GIP / L-GLUT COMPLEX (2L4T)
MMP-12 (2POJ)
STT3P (2LGZ)
GIP (2L4S)
Clemson CI Symposium - Feb 13, 2013
PERTURBATION OF MMP12 BY Triple Helical Peptide AND THE MAPPING
OF THEM ON THE STRUCTURE OF MMP12.
0 .0 5
K d = 30 ± 6 M
H112
Binding Isotherm
G221
0 .0 4
L147
T205
K148
Y240
L224
0 .0 3
H
  (p p m )
Y113
T210
V243
0 .0 2
0 .0 1
0 .0 0
0 .0 0 0 0
0 .0 0 0 1
0 .0 0 0 2
0 .0 0 0 3
[ a 1 (V )T H P ] (M )
1
15
Chem ical S hift P ertu rb atio n of H - N N M R
Correlatio n S pectru m for Finding Bindin g Site
15 N
1
H
Clemson CI Symposium - Feb 13, 2013
Bhaskaran et al. J Biol Chem.(2008)
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
Clemson CI Symposium - Feb 13, 2013
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
Clemson CI Symposium - Feb 13, 2013
Palmier, Bhaskaran et al. J. Biol. Chem. (2010)
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
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. thermophilus Proteins
Dr. P. Karthe, University of Madras, Chennai, India :
NMR structure of Serine glutamate repeat A, a surface adhesin
Clemson CI Symposium - Feb 13, 2013
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. Homayoun Valafar, 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. Esmaeil Jabbari, 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
ACKNOWLEDGEMENT
Mr. Raghav Nagampalli (Fulbright Scholar)
Dr. Angela Peters (Claflin)
Dr. Verlie Tisdale (Claflin)
School of Natural Sciences and Mathematics, Claflin
SC INBRE
NASA EPSCoR Space Grant.
TRUSTWORTHINESS
RESPECT
RESPONSIBILITY
FAIRNESS
CITIZENSHIP
CARING
Clemson CI Symposium - Feb 13, 2013
THANK YOU ALL
Clemson CI Symposium - Feb 13, 2013