The forces which control the formation of secondary and
tertiary structure in biological macromolecules are understood
only in a very general sense. It is as yet impossible to
predict the three-dimensional structure of a globular protein
based only on sequence information. The complexity of the
problem is the result of the fact that the tertiary structure
of a protein represents a compromise between a large number
of interactions involving the protein and solvent which,
though individually weak, act cooperatively to give rise
to the observed structure. We are using several different
approaches to deconvolute this complex problem and improve
our understanding of the forces determining protein structure.
We
have used multi-dimensional and multinuclear NMR methods
to determine the structure of a globular electron-transfer
protein, putidaredoxin (or Pdx). Pdx contains a 2-Fe 2-S
cluster and is archetypal of a group of small proteins which
act as selective electron shuttles from NADH-dependent flavoproteins
to P-450 cytochromes. Pdx is the first of these redoxins
for which a structure has been determined, and we are now
in a good position to begin answering questions about the
recognition and binding of a metal cluster by a nascent
ferredoxin. Why is the metal required for folding? What
form do the components of the cluster take in solution?
How selective is the binding of a particular metal? Using
NMR, mutagenesis and physical methods, we are continuing
our investigation of the structure, dynamics and folding
of Pdx and related proteins.
At the other end of the scale, we are attempting to quantitate
the relative free energies of amino acid side chain-side
chain interactions using what we call "side chain mimic"
HPLC stationary phases. These are simply solid HPLC supports
such as silica gel to which are chemically bound compounds
which mimic the side chains of naturally occurring amino
acids. Chromatographic retention times can be related to
relative free energies of adsorption onto the stationary
phase, so the interaction between peptide analytes and the
side chain bound to the solid support are reflected by chromatographic
retention. Since to a first approximation, the only way
in which the naturally occurring amino acids differ (except
proline) is in their side chains; different chromatographic
retentions for different amino acid derivatives should reflect
only the differences in interaction between the support-bound
side chains and the peptides being analyzed. The data which
we have obtained in this fashion are being used to develop
Monte Carlo computer simulations which we hope will further
clarify the rules which govern protein folding.
Selected Publications:
Bo OuYang, Susan Sondej Pochapsky, Marina Dang, and Thomas C. Pochapsky (2008) A Functional Proline Switch in Cytochrome P450cam. Structure. 2008 May 7; 16(5).
OuYang B, Pochapsky SS, Pagani GM, Pochapsky TC. (2006)
Specific effects of potassium ion binding on wild-type and
L358P cytochrome P450cam. Biochemistry. 2006 Dec
5;45(48):14379-88. [abstract]
Ju T, Goldsmith RB, Chai SC, Maroney MJ, Pochapsky SS,
Pochapsky TC. (2006) One protein, two enzymes revisited:
a structural entropy switch interconverts the two isoforms
of acireductone dioxygenase. J Mol Biol. 2006 Nov
3;363(4):823-34. [abstract]
Rui L, Pochapsky SS, Pochapsky TC. (2006) Comparison of
the complexes formed by cytochrome P450cam with cytochrome
b5 and putidaredoxin, two effectors of camphor hydroxylase
activity.Biochemistry. 2006 Mar 28;45(12):3887-97.
[abstract]
Pochapsky TC, Pochapsky SS, Ju T, Hoefler C, Liang J. (2006)
A refined model for the structure of acireductone dioxygenase
from Klebsiella ATCC 8724 incorporating residual dipolar
couplings. J Biomol NMR. 2006 Feb;34(2):117-27. [abstract]
Sauter M, Lorbiecke R, Ouyang B, Pochapsky TC, Rzewuski
G. (2005) The immediate-early ethylene response gene OsARD1
encodes an acireductone dioxygenase involved in recycling
of the ethylene precursor S-adenosylmethionine. Plant J.
2005 Dec;44(5):718-29. [abstract]
Wei J.Y., Pochapsky T.C., and Pochapsky S.S. (2005) Detection
of a High-Barrier Conformational Change in the Active Site
of Cytochrome P450cam upon Binding of Putidaredoxin.
Journal of the American Chemical Society, 127:
6974-6976.
Zhang Y., Heinsen M.H., Kostic M., Pagani G.M., Riera T.V.,
Perovic .I, Hedstrom L., Snider B.B., Pochapsky T.C. (2004)
Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane,
an acyclic intermediate in the methionine salvage pathway:
a new preparation and characterization of activity with
E1 enolase/phosphatase from Klebsiella oxytoca. Bioorg
Med Chem. 12:3847-55. [abstract]
Kostic M., Bernhardt R, Pochapsky TC. (2003) A conserved
histidine in vertebrate-type ferredoxins is critical for
redox-dependent dynamics. Biochemistry. 42:8171-82.
[abstract]
Pochapsky SS, Pochapsky TC, Wei JW. (2003) A model for
effector activity in a highly specific biological electron
transfer complex: the cytochrome P450(cam)-putidaredoxin
couple. Biochemistry. 42:5649-56. [abstract]
Pochapsky TC, Pochapsky SS, Ju T, Mo H, Al-Mjeni F, Maroney
MJ. (2002) Modeling and experiment yields the structure
of acireductone dioxygenase from Klebsiella pneumoniae.
Nat Struct Biol. 9:966-72 [abstract].
M. Kostic, T. C. Pochapsky and S. S. Pochapsky. (2002)
"Rapid Recycle 13C, 15N and 13C, 13C' Heteronuclear and
Homonuclear Multiple Quantum Coherence Detection for Resonance
Assignments in Paramagnetic Proteins: Example of Ni+2-Containing
Acireductone Dioxygenase (ARD)", J. Am. Chem. Soc.,
124(31):9054-9055 [abstract].
M. Kostic, S. S. Pochapsky, T. C. Pochapsky, J. Obenauer,
H. Mo, G. M. Pagani and R. Pejchal. (2002) "Comparison of
functional domains in vertebrate-type ferredoxins" Biochemistry
41: 5978-5989. [abstract]
Al-Mjeni, T. Ju, T. C. Pochapsky, and M. J. Maroney. (2002)
"XAS Investigation of the Structure and Function of Ni in
Acireductone Dioxygenase" F. Biochemistry 41: 6761-6769.
R. Mukhopadhyay, L. L. Wong, K. K. Lo, T. Pochapsky and
H. A. O. Hill. (2002) "A Molecular Level Study of Complex
Formation between Putidaredoxin and Cytochrome P450 by Scanning
Tunneling Microscopy." Physical Chemistry Chemical Physics
641-646.
T. C. Pochapsky and S. S. Pochapsky. (2001) "Nuclear magnetic
resonance as a tool in drug discovery, metabolism and disposition."
Curr. Top. Med. Chem. 1: 427-441. [abstract]
T. C. Pochapsky, M. Kostic, N. Jain and R. Pejchal. (2001)
"Redox-dependent conformational selection in a Cys4Fe2S2
ferredoxin" Biochemistry 19: 5602-5614. [abstract].
Y. Dai, T. C. Pochapsky and R. H. Abeles. (2001) "Mechanistic
studies of two dioxygenases in the methionine salvage pathway
of Klebsiella pneumoniae" Biochemistry, 40: 6379-6387.
[abstract]
T. C. Pochapsky and C. Hofstetter. (2000) "BF4- as a probe
for ion pair solution structure using interionic one- and
two-dimensional 19F{1H} NOEs" Magn. Reson. Chem.
38: 90-94.
Pereira de Araujo AF, Pochapsky TC, Joughin B. (1999) Thermodynamics
of interactions between amino acid side chains: experimental
differentiation of aromatic-aromatic, aromatic-aliphatic,
and aliphatic-aliphatic side-chain interactions in water.
Biophys J. 76: 2319-28. [abstract]
Mo H, Pochapsky SS, Pochapsky TC. (1999) A model for the
solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin
from Pseudomonas. Biochemistry. 38: 5666-75. PDF
version. [abstract]
Pochapsky TC, Jain NU, Kuti M, Lyons TA, Heymont J. (1999)
A refined model for the solution structure of oxidized putidaredoxin.
Biochemistry. 38: 4681-90.
PDF
version. [abstract]
Jain NU, Pochapsky TC. (1999) A new assignment strategy
for the hyperfine-shifted 13C and 15N resonances in Fe2S2
ferredoxins. Biochem Biophys Res Commun. 258:
54-9. [abstract]
Arico-Muendel CC, Patera A, Pochapsky TC, Kuti M, Wolfson
AJ. (1999) Solution structure and dynamics of a serpin reactive
site loop using interleukin 1beta as a presentation scaffold.
Protein Eng. 12:189-202. [abstract]
Mo H, Dai Y, Pochapsky SS, Pochapsky TC. (1999) 1H, 13C
and 15N NMR assignments for a carbon monoxide generating
metalloenzyme from Klebsiella pneumoniae. J Biomol NMR.
14: 287-8.
T. C. Pochapsky. (1999) "Designed molecular recognition:
A commentary on possible design elements". Enantiomer
4: 437-444.
T. C. Pochapsky, C. Hofstetter and P. S. Wilkinson. (1999)
"NMR structure determination of ion pairs derived from quinine:
A model for templating in asymmetric phase transfer reductions
by BH4- with implications for rational design of phase transfer
catalysts" J. Org. Chem. 64: 8794-8800.
Pochapsky TC, Kuti M, Kazanis S. (1998) The solution structure
of a gallium-substituted putidaredoxin mutant: GaPdx C85S.
J Biomol NMR. 12: 407-15. [abstract]
N. Jain and T. C. Pochapsky, (1998). J. Am. Chem. Soc.
120: 12984-12985.
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