NMR - Major Users and Research Projects
Jason Chesney (BCC)
Our laboratory is using an NMR-based metabolomics approach to understand the
regulation of glucose metabolism in cancer cells, via the activity of iPFK-2.
We are using isotopomer analyses in conjunction with molecular biology approaches
(e.g. siRNA) to modulate specific enzyme activities to further test the Warburg
hypothesis and examine the fate of glucose carbon in transformed versus untransformed
cell types.
Selected References:
Chesney J, Mitchell R, Benigni F, Bacher M, Spiegel L, Al-Abed Y, Han JH, Metz
C, Bucala R. (1999) An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich
instability element: Role in tumor cell glycolysis and the Warburg effect. Proc. Natl.
Acad. Sci. 96, 3047-3052
Atsumi T, Chesney J, Metz C, Leng L, Donnelly S, Makita Z, Mitchell R, Bucala
R. (2002) High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
(iPFK-2;
PFKFB3) in human cancers Cancer Research 62, 5881-5887
Teresa Fan (Dept. Chemistry UofL)
Our laboratory is using NMR methods in three major areas. (1) metabolomics
characterization of cells and their responses to mutations and environmental
insults and the mechanisms of Se compounds in these processes. (2)
Structural characterization of the SECIS 3’-RNA species in regulation
of SeCys incorporation into critical enzymes. (3) Analysis of soil organic
matter chemistry and remediation against environmental toxicants. These projects
all make use of the spectral editing capabilities of partially labeled samples. For
example, isotopomer analysis is carried out using a combination of 2D 13C-1H
edited experiments including HSQC, HCCHTOCSY and HSQC-TOCSY. We also
make use of 77Se NMR for analyzing selenium metabolites involved in anticancer
activity
Selected References:
T.W-M Fan (1996) Metabolite profiling by one- and two-dimensional NMR analysis
of complex mixtures. Prog. NMR Spectrosc. 28,
161-219
Fan, T. W.-M., R. M. Higashi and A. N. Lane. (2000) “Chemical Characterization
of a Chelator-Treated Soil Humate by Solution-State Multinuclear Two-Dimensional NMR with FTIR and
Pyrolysis-GCMS”, Environmental Science and Technology 34, 1636-1646
Fan, T. W-M., Higashi, R.M.,. & Lane, A.N (2004) The Promise of Metabolomics
in Cancer Molecular Therapeutics. Current Opin. Molec. Ther. In press
Ramos, A., Lane, A.N., Hollingworth, D. & Fan, T.W-M.(2004) NMR determination
of the secondary structure of the selenocysteine insertion element. Nucl. Acids
Res. 32, 1746-1755
Robert Gray (Dept. Biochem. UofL)
Biochemistry of APRin. Our laboratory is using a variety of
biochemical and biophysical methods to characterize the structure, stability
and interactions of the alkaline proteinase inhibitor (APRin) from Pseudomonas
aeruginosa. This proteinis an 11.5-kDa, high affinity, high specificity
inhibitor of the serralysin class of zinc-dependent proteinases secreted
by several Gram-negative bacteria (1-3).
We are using high resolution NMR to determine the solution conformation
and dynamics of APRin. With 15N/13C labeled protein, most of the backbone
and side chain assignments have been obtained (4). Analysis of the chemical
shifts and NOEs reveals the secondary structure, and provides information about
the dynamics on the N-terminal region essential for protease inhibition. The
solution structure is currently being calculated. This will assist understanding
the stability and binding mechanism of the inhibitor to its cognate metalloproteinase
presently under investigation.
Selected References:
Feltzer, R. E., Gray, R. D., Dean, W. L., Pierce, W. M. Jr. (2000) Alkaline
Proteinase Inhibitor of Pseudomonas aeruginosa. Interaction
of Native and N-terminally Truncated Inhibitor Proteins with Pseudomonas Metalloproteinases. J.
Biol. Chem. 275, 21002-21009
Feltzer, R. E., Trent, J. O., and Gray, R. D. (2003) Alkaline Proteinase
Inhibitor of Pseudomonas aeruginosa: A Mutational and Molecular Dynamics
Study of The Role of N-Terminal Residues in the Inhibition of Pseudomonas Alkaline
Proteinase. J. Biol. Chem., 278, 25952-25957
Hege, T., Feltzer, R. E., Gray, R. D., and Baumann, U. (2001) Crystal Structure
of a Complex Between Pseudomonas aeruginosa Alkaline Protease and
its Cognate Inhibitor. J. Biol. Chem. 276, 35087-35092
Sengodagounder Arumugam, Robert D. Gray and Andrew N. Lane,
1H, 15N and 13C assignments of the alkaline proteinase inhibitor APRin from Pseudomonas
aeruginosa. J. Biomolec NMR. Submitted
Andrew Lane (BCC)
My laboratory is concerned primarily with understanding the functional consequences
of molecular interactions, especially protein-nucleic acid and protein-protein
interactions, which often are the first step in starting a cascade of gene
expression. We use high-resolution NMR methods to analyze the solution conformations
and dynamics of macromolecules and more importantly the changes in these
properties due to complex formation. In addition to our interests in detailed
molecular structures and dynamics, we collaborate with a variety of cell
and molecular biologists on the regulation of metabolic process that occur
in cancer cells. This also uses NMR methods in concert with a variety of
analytical techniques such as GC-MS, transcription and proteomics. We
are therefore also involved in methods development to solve new problems
as they arise.
Selected References:
Lane, A.N., Kelly, G., Ramos, A. & Frenkiel, T.A. (2001) Determining binding
sites in protein-nucleic acid complexes by cross-saturation. J. Biomolec. NMR 21, 127-139
Nair, M., McIntosh, P.B., Frenkiel, T.A., Kelly, G., Taylor,I.A., Smerdon, S.J., & Lane A.N. (2003)
NMR Structure of the DNA-binding Domain of the Cell Cycle protein, Mbp1 from Saccharomyces
cerevisiae. Biochemistry, 42 1266 –1273
Gyi, J.I., Gao, D., Conn, G.L., Trent, J.O., Brown, T. & Lane, A.N. (2003) The solution structure of a DNA·RNA duplex
containing 5-propynyl U and C; comparison with 5-Me modifications. Nucl. Acids Res 31,
2683-2693.
Ramos, A., Lane, A.N., Hollingworth, D. & Fan, T.W-M.(2004) NMR determination
of the secondary structure of the selenocysteine insertion element. Nucl. Acids Res. 32,
1746-1755
Fan, T. W-M., Higashi, R.M.,. & Lane, A.N (2004) The Promise of Metabolomics in Cancer Molecular Therapeutics.
Current Opin. Molec. Ther. 6:584-592
Booth, J., Brown, T., Vadhia, S.J., Lack, O., Cummins,W.J., Trent,
J.O., & Lane A.N. .(2005) Determining the origin of
the stabilization of DNA by 5- aminopropynylation of pyrimidines. Biochemistry.
44,4710-4719
Muriel Maurer (Dept. Chemistry UofL)
http://www.louisville.edu/a-s/chemistry/faculty/mcm/prf.htm
The Maurer group uses a combination of protein chemistry mass spectrometry
and high resolution NMR to study molecular recognition and mecgan9osn in blood
coagulation and related processes. Our focus is on the mechanism of thrombin
activation of factor XIII.
A combination of kinetic studies, NMR, hydrogen/deuterium exchange coupled
with MALDI-TOF mass spectrometry, and molecular modeling is being employed.
Selected References:
Turner, B.T., Sabo, T.M., Wilding, D., Maurer, M.C. (2004) “Mapping
of Factor XIII Solvent Accessibility as a Function of Activation State Using
Chemical Modification Methods”, Biochemistry 43,
9755-65.
Isetti, G., Maurer, M.C. (2004) “N-terminal Truncation of Factor XIII
Activation Peptides (28-37) V34 and V34L does not Hinder Ability to Interact
with Thrombin”, Biochemistry 43, 4150-4159.
Trumbo, T.A., Maurer, M.C. (2003) “V34I and V34A Substitutions Within
the Factor XIII Activation Peptide Segment (28-41) Affect Interactions
With the Thrombin Active Site”, Thrombosis and Haemostasis 89,
647-653.
Turner, B.T. Jr, Maurer, M.C. (2002) “Evaluating the Roles of Thrombin
and Calcium in the Activation of Coagulation Factor XIII Using H/D Exchange
and MALDI-TOF MS” Biochemistry 41, 7947-7954.
Anne-Frances Miller (Dept. Chemistry UK)
http://www.chem.uky.edu/research/miller/
The Miller research group uses a variety of spectroscopic techniques to address mechanisms
of catalysis in the redox/metal dependent enzymes in the following
three areas.
- Superoxide Dismutases. The Fe-SOD and Mn-SOD are being explored.
EPR and NMR are used to explore the details of the mutant structures of these
enzymes.
- Nitroreductase. Multidimensional NMR is applied to evaluate the mechanism
of NR for the clean-up of the left-over of bombs.
- Peptide Deformylase. This is a novel drug target. The details of
the catalytic mechanism and the interaction of the effective inhibitors with
PDF are explored by spectroscopic pH titration using UV-visible spectrophotometer
and NMR.
Selected References:
Miller, Anne-Frances. Superoxide dismutases: active sites that save, but a protein that kills. Current Opinion in Chemical
Biology (2004), 8(2), 162-168.
Jackson, Timothy A.; Yikilmaz, Emine; Miller, Anne-Frances; Brunold, Thomas C. Spectroscopic and computational study of a non-heme iron {Fe-NO}7 system: Exploring the
geometric and electronic structures of the nitrosyl adduct of iron superoxide dismutase.J.
Am. Chem. Soc. (2003), 125(27), 8348-8363.
Walsh, Joseph D.; Miller, Anne-Frances. NMR Shieldings and Electron
Correlation Reveal
Remarkable Behavior on the Part
of the Flavin N5 Reactive Center. J. Phys. Chem. B
(2003), 107(3), 854-863.
Koder, Ronald L.; Haynes, Chad A.; Rodgers, Michael E.; Rodgers, David W.;
Miller, Anne-Frances. Flavin Thermodynamics
Explain the Oxygen Insensitivity of Enteric
Nitroreductases.Biochemistry (2002), 41(48), 14197-14205.
Peter Spielmann (Dept. Biochemistry, UK)
http://www.mc.uky.edu/biochemistry/Department/faculty/Spielmann/
We study and characterize the structural and dynamic features of damaged and
undamaged DNA. These studies are used to elucidate the structural features
for initial substrate recognition for the three main DNA repair pathways. Identification
of these conformations may ultimately lead to the design of a new class of
compounds to inhibit DNA repair and thereby enhance the effectiveness of DNA
damaging antineoplastic therapeutics. Formation of carcinogen-DNA adducts alters
the conformation and dynamics of the polymer in such a way that DNA conformations
not accessible to undamaged DNA become available. These abnormal conformations
may be the recognition motif for nucleotide excision repair (NER) and methyl
directed mismatch repair (MMR). We have determined that covalent DNA damage
alters both the structure and local high-frequency dynamics of the polymer
for a preliminary set of molecules. These data suggest that there exist DNA
conformations not easily accessible to undamaged DNA that become populated
(or accessible) to damaged DNA. We use NMR techniques to obtain experimental
data to describe the three-dimensional structure and internal dynamics of site-specifically
damaged DNA and its undamaged counterpart. We have developed restrained molecular
dynamics (rMD) computational procedures that provide good agreement between
experimentally determined dynamic properties and dynamic features revealed
by the rMD simulations.
Selected References:
Isaacs, R. J. and Spielmann, H. P. (2004) "A Model for Initial DNA Lesion Recognition by NER
and MMR Based on Local Conformational Flexibility" DNA Repair 3, 455-64.
Isaacs, R. J. and Spielmann, H. P. (2004) "Insight into G-T Mismatch Recognition Using
Molecular Dynamics with Time-Averaged Restraints Derived from NMR Spectroscopy" J. Am. Chem. Soc. 126, 583-90.
Isaacs, R. J., Rayens, W. T. and Spielmann, H. P. (2002) "Structural
Differences in the NOE-Derived Structure of G-T Mismatched DNA Relative to Normal DNA Are Correlated With
Differences in 13C Relaxation-Based Internal Dynamics" J. Mol. Biol 319 191-207.
Isaacs, R. J. and Spielmann, H. P. (2001) "NMR Evidence for Mechanical
Coupling of Phosphate BI-BII Transitions With Deoxyribose Conformational Exchange in DNA"
J. Mol. Biol. 311, 149-160.
Isaacs, R. J. and Spielmann, H. P. (2001) "Relationship of DNA Structure to Internal Dynamics:
Correlation of Helical Parameters from NOE Based NMR Solution Structures of d(GCGTACGC)2 and d(CGCTAGCG)2 with
13C Order Parameters Implies Conformational Coupling in Dinucleotide Units" J. Mol. Biol. 307,
525-540.
Spielmann, H. P. (1998) "Dynamics of a Bis-intercalator DNA Complex
by 1H Detected Natural Abundance 13C NMR Spectroscopy" Biochemistry 37, 16863-16876.
Spielmann, H. P. (1998) "Dynamics in Psoralen Damaged DNA by 1H
Detected Natural Abundance 13C NMR Spectroscopy" Biochemistry 37, 5426 -5438.
John Trent (BCC): Nucleolin and G-quartet structures
www.browncancercenter.org/researchweb/trent/trent.html
We have expressed the RNA binding domains 1 and 2 of human nucleolin
with the RGG tail in E. coli. The construct binds to a G-rich oligonucleotide
aptamer DNA anticancer molecule, and is being further characterized by thermodynamic
and NMR methods. The construct is being examined at high field by triple resonance
methods using the 15N/13C labeled protein. The interaction surface with the
DNA is being mapped using 15N labeled protein and a combination of HSQC-based
chemical shift mapping and cross-saturation spectroscopy from the DNA imino
protons. The combination of the protein structure, the residues involved in
the protein-DNA interaction and the structure of the quartet will allow detailed
modeling of the interaction, and provide the basis for understanding specific
mutations on the functional properties. Isotopic labeling of the G-quartet
by chemical synthesis is being used to verify features of the model.
Selected References:
Bates, PJ, Kahlon, JB, Thomas, SD, Trent, JO & Miller, DM (1999) J.
Biol. Chem. 274, 26369-77
Dapic V, Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent JO, Bates
PJ (2003) Biophysical and biological properties of quadruplex oligodeoxyribonucleotides Nucleic
Acids Research 31, 2097-2107
Richard Wittebort (Dept. Chemistry UofL)
Our laboratory uses solid state NMR techniques to probe structure, dynamics
and hydration in oligo and polypeptides. We have been measuring the chemical
shielding tensors in single crystals of 13C and 15N labeled peptides. Recently
we have been using ultrahigh field solid state NMR (at 18.8 T) to measure disorder
and dynamics in unstructured proteins such as elastin. We are also expressing
recombinant collagen peptides that are being studied by both solution and solid
state NMR methods.
Selected References:
Chekmenev, E.Y., Xu, R.Z., Mashuta, M.S. and Wittebort, R.J., “Clycyl
Ca Chemical Shielding in Tripeptides: Measurement by Solid-State NMR and Correlation
with X-ray Structure and Theory”, J. Am. Chem. Soc., 124,
11894-11899 (2002)
Zhang, Q., E. Y. Chekmenev and R. J. Wittebort,"17O Quadrupole Coupling
and Chemical Shielding Tensors in an H-bonded Carboxyl Group: Oxalic Acid", J.
Am. Chem. Soc. 125, 9140-9146 (2003)
Cheruzel, L.E., Pometun, M.S., Cecil, M.R., Wittebort, R.J. and Buchanan, R.M., “Structures
and Solid State Dynamics of One-Dimensional Water Chains Stabilized by Imidazole
Channels”, Angewandte Chem, Int. Ed., 42, 5452-5455
(2003)
Chekmenev, E.Y., Zhang, Q., Waddell, K.W., Mashuta, M.S. and Wittebort, R.J. “15N
Chemical Shielding in Glycyl Tripeptides: Measurement by Solid-State NMR and
Correlation with X-ray Structure”, J. Am. Chem. Soc, 126,
379-384 (2004)
Pometun, M. S., Chekmenev, E.Y. and Wittebort, R.J. “Quantitative Observation
of Backbone Disorder in Native Elastin”, J. Biol. Chem, 279,
7982-7987 (2004)
Other users:
J. Eaton (BCC): Oxygen-tolerant metabolism in HeLa cells
K. Ramos (Dept. Biochemistry): Metabolic profiling during nephrogenesis
R. Valdes (Dept. Pathology and Clinical Medicine): Structural identification of digoxin-like immunoreactive factors (DLIFs)