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FRISC: The Faculty Research Interests Science Comparator |
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Assistant Professor of Biochemistry
Biological Chemistry
Molecular Biophysics
Phone: (214) 648-8916
FAX: (214) 648-8947
Email: kgardn@biochem.swmed.edu
Home Page: Gardner Lab
Our laboratory uses solution nuclear magnetic resonance (NMR) methods to study the
structure and dynamics of protein/ligand and protein/protein complexes involved in
regulating eukaryotic transcription.
Studies such as these have been made possible by the rapid evolution of NMR-based tools
to study macromolecular structure. Until recently, NMR methods provided structures
of only relatively small proteins under 15 kDa molecular weight. However, the
development of stable isotope labeling, multidimensional NMR pulse sequences and
computational methods now enables solution NMR approaches to obtain high-resolution
structures of 30-40 kDa protein systems. One can also use these methods to get
useful structural information on even larger proteins and complexes, as will be of
particular use to our group as we study macromolecular assemblies involved in
transcriptional regulation.
As mentioned above, methods for effectively labeling proteins with stable isotopes have
been critical to many of these advances. Of particular utility for our studies are
schemes to produce proteins uniformly labeled with isotopes of nitrogen ( 15 N), carbon ( 13 C) and hydrogen ( 2 H). Using proteins
labeled in this manner, I was able to assign the backbone and many of the sidechain
chemical shifts of a 42 kDa maltose binding protein/carbohydrate complex (Gardner et al .,
1998). For proteins over 20kDa, such studies are only made possible by the
improvements in spectral sensitivity and resolution offered by 2 H labeling.
Unfortunately, these benefits have a significant cost: uniform 2 H labeling drastically reduces
the number of interproton NOE-based distance restraints one can measure among the
remaining protons. As these restraints constitute the majority of data used to
generate NMR-derived structures, reducing their number and diversity in this way
significantly worsens the precision and accuracy of structures available from fully
deuterated proteins. As such, I have developed chemical and biochemical approaches
to generate highly deuterated proteins that contain a small number of highly protonated
sites, typically at the methyl groups of Val, Leu and Ile (Gardner & Kay, 1997; Goto et
al. , 1999). Proteins produced in this manner retain many of the benefits of
deuteration, while methyl-amide and methyl-methyl NOEs provide an ensemble of distance
restraints significantly larger than those previously available, allowing the generation
of medium resolution structures of proteins over 40 kDa using exclusively NMR-based data.
Selected Publications:
Goto, N.K., Gardner, K.H., Mueller G.A., Willis, R.C. and Kay, L.E. (1999) A
robust and cost-effective method for the production of Val, Leu and Ile( d 1) methyl-protonated 15 N, 13 C, 2 H-labeled
proteins. J. Biomol. NMR , 13: 369-374.
Gardner, K.H. and Kay, L.E. (1998) The use of 2 H, 13 C, 15 N multidimensional NMR to study the structure and dynamics of proteins . Ann.
Rev. Biophys. Biomol. Struct. , 27: 357-406.
Gardner, K.H., Zhang, X., Gehring, K. and Kay, L.E. (1998) Solution
NMR studies of a 42 kDa maltose binding protein/ b -cyclodextrin complex: chemical shift assignments and analysis , J. Am.
Chem. Soc. , 120: 11738-11748.
Zwahlen, C., Gardner, K.H., Sarma, S.P., Horita, D.A., Byrd, R.A. and Kay, L.E. (1998) An
NMR experiment for measuring methyl-methyl NOEs in 13 C labeled proteins with high resolution , J. Am. Chem. Soc. , 120:
7617-7625.
Gardner, K.H. and Kay, L.E. (1997) Production
and incorporation of 15 N, 13 C,
2 H ( 1 H- d 1 methyl) isoleucine into proteins for multidimensional NMR studies . J.
Am. Chem. Soc. , 119: 7599-7600.
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Last updated: 17 Nov 2000
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