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:

Redox-dependent dynamics in cytochrome P450cam. Pochapsky SS, Dang M, OuYang B, Simorellis AK, Pochapsky TC. Biochemistry. 2009 May 26;48(20):4254-61. [abstract]

Structural and dynamic implications of an effector-induced backbone amide cis-trans isomerization in cytochrome P450cam. Asciutto EK, Madura JD, Pochapsky SS, OuYang B, Pochapsky TC. J Mol Biol. 2009 May 15;388(4):801-14. [abstract]

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation.  Zhang W, Pochapsky SS, Pochapsky TC, Jain NU. J Mol Biol. 2008 Dec 12;384(2):349-63. [abstract]

A Functional Proline Switch in Cytochrome P450cam. Bo OuYang, Susan Sondej Pochapsky, Marina Dang, and Thomas C. Pochapsky (2008) Structure. 2008 May 7; 16(5). [abstract]

Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam. OuYang B, Pochapsky SS, Pagani GM, Pochapsky TC. (2006) Biochemistry. 2006 Dec 5;45(48):14379-88. [abstract]

One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase. Ju T, Goldsmith RB, Chai SC, Maroney MJ, Pochapsky SS, Pochapsky TC. (2006) J Mol Biol. 2006 Nov 3;363(4):823-34. [abstract]

Comparison of the complexes formed by cytochrome P450cam with cytochrome b5 and putidaredoxin, two effectors of camphor hydroxylase activity. Rui L, Pochapsky SS, Pochapsky TC. (2006) Biochemistry. 2006 Mar 28;45(12):3887-97. [abstract]

A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings. Pochapsky TC, Pochapsky SS, Ju T, Hoefler C, Liang J. (2006) J Biomol NMR. 2006 Feb;34(2):117-27. [abstract]

The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine. Sauter M, Lorbiecke R, Ouyang B, Pochapsky TC, Rzewuski G. (2005) Plant J. 2005 Dec;44(5):718-29. [abstract]

Detection of a High-Barrier Conformational Change in the Active Site of Cytochrome P450cam upon Binding of Putidaredoxin. Wei J.Y., Pochapsky T.C., and Pochapsky S.S. (2005) Journal of the American Chemical Society, 127: 6974-6976.

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. Zhang Y., Heinsen M.H., Kostic M., Pagani G.M., Riera T.V., Perovic .I, Hedstrom L., Snider B.B., Pochapsky T.C. (2004) Bioorg Med Chem. 12:3847-55. [abstract]

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics. Kostic M., Bernhardt R, Pochapsky TC. (2003) Biochemistry. 42:8171-82. [abstract]

A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple. Pochapsky SS, Pochapsky TC, Wei JW. (2003) Biochemistry. 42:5649-56. [abstract]

Modeling and experiment yields the structure of acireductone dioxygenase from Klebsiella pneumoniae. Pochapsky TC, Pochapsky SS, Ju T, Mo H, Al-Mjeni F, Maroney MJ. (2002) Nat Struct Biol. 9:966-72 [abstract].

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). Kostic M, Pochapsky TC, Pochapsky SS. (2002) J. Am. Chem. Soc., 124(31):9054-9055 [abstract].

Comparison of functional domains in vertebrate-type ferredoxins. Kostic M, Pochapsky SS, Pochapsky TC, Obenauer J, Mo H, Pagani GM and Pejchal R. (2002) Biochemistry 41: 5978-5989. [abstract]

XAS Investigation of the Structure and Function of Ni in Acireductone Dioxygenase. Al-Mjeni F, Ju T, Pochapsky TC, and Maroney MJ. (2002) Biochemistry 41: 6761-6769.

A Molecular Level Study of Complex Formation between Putidaredoxin and Cytochrome P450 by Scanning Tunneling Microscopy. Mukhopadhyay R, Wong LL, Lo KK, Pochapsky T and Hill HA. (2002) Physical Chemistry Chemical Physics 641-646.

Nuclear magnetic resonance as a tool in drug discovery, metabolism and disposition. Pochapsky TC and Pochapsky SS. (2001) Curr. Top. Med. Chem. 1: 427-441. [abstract]

Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin. Pochapsky TC, Kostic M, Jain N, Pejchal R. (2001) Biochemistry 19: 5602-5614. [abstract].

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae. Dai Y, Pochapsky TC and Abeles RH. (2001) Biochemistry, 40: 6379-6387. [abstract]

BF4- as a probe for ion pair solution structure using interionic one- and two-dimensional 19F{1H} NOEs. Pochapsky TC and Hofstetter C. (2000) 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]

A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas. Mo H, Pochapsky SS, Pochapsky TC. (1999) Biochemistry. 38: 5666-75. PDF version. [abstract]

A refined model for the solution structure of oxidized putidaredoxin. Pochapsky TC, Jain NU, Kuti M, Lyons TA, Heymont J. (1999)Biochemistry. 38: 4681-90. PDF version. [abstract]

A new assignment strategy for the hyperfine-shifted 13C and 15N resonances in Fe2S2 ferredoxins. Jain NU, Pochapsky TC. (1999) Biochem Biophys Res Commun. 258: 54-9. [abstract]

Solution structure and dynamics of a serpin reactive site loop using interleukin 1beta as a presentation scaffold. Arico-Muendel CC, Patera A, Pochapsky TC, Kuti M, Wolfson AJ. (1999) Protein Eng. 12:189-202. [abstract]

1H, 13C and 15N NMR assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae. Mo H, Dai Y, Pochapsky SS, Pochapsky TC. (1999) J Biomol NMR. 14: 287-8.

Designed molecular recognition: A commentary on possible design elements. Pochapsky TC. (1999) . Enantiomer 4: 437-444.

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. Pochapsky TC, Hofstetter C and Wilkinson PS. (1999) J. Org. Chem. 64: 8794-8800.

 

 

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Last update: July 28, 2009.