The largest project in our laboratory deals with structure and function of cytochrome P450 monooxygenases in bacterial and mammalian cells. The cytochrome P450s are a large family of enzymes that are critical in a wide variety of metabolic processes, including drug metabolism, carcinogen activation, steroid biosynthesis and xenobiotic clearance. Using multidimensional NMR, molecular biology, organic synthesis, computational methods and biochemical assays, we are unraveling the complex structure-function relationships in cytochrome P450cam (CYP101), the archetype of the P450 family. In particular, we are interested in the role of effector binding on P450 function. It is well-known that CYP101 requires the presence of the iron-sulfur protein putidaredoxin (Pdx) in order to turn over substrate. NMR methods have revealed that a series of complex changes take place in the CYP101 structure upon binding of Pdx, and this has led us to propose a model for how these changes occur and why they are critical to enzymatic activity (64,68,71).Currently, we are extending the methodology and expertise that we have developed with CYP101 to the important class of P450s that are involved in drug metabolism.
The complex between Pdx and CYP101 shown in the Figure 1 was generated by our group using molecular dynamics simulations (37). We determined the solution structure of Pdx using NMR methods. Pdx was the first protein of its class for which a structure was determined, and the first metalloprotein of any sort for which a structure was determined exclusively by NMR without reference to a homologous crystal structure. (25,31,48) The interaction between Pdx and CYP101 is modulated by the redox state of Pdx, and we characterized structural and dynamic differences between the oxidized and reduced forms of Pdx using 1H-15N correlated spectroscopy. (32,35) We proposed a model in which reduced Pdx, which binds more tightly to CYP101 than the oxidized form, occupies a subset of conformations that match the binding surface of CYP101 more precisely than the oxidized form, and showed by mutagenesis that we could slow the interchange between these conformations so that they could be observed discretely by 2D NMR (55) .
We used NMR and site-directed mutagenesis to determine the origins of functional differences between Pdx and related bacterial and mammalian ferredoxins, including terpredoxin (Tdx), which is the redox partner of cytochrome P-450terp, and the human ferredoxin adrenodoxin (Adx), which transfers electrons to cytochromes P450 in the steroidogenic pathways in humans. These three ferredoxins all have similar tertiary structures (47), but show remarkable specificity for interacting only with their own cognate P450s. We have identified a number of sequence differences between the three proteins that may be important in determining the low cross-reactivity of the ferredoxins. (65)
One of the biggest difficulties facing NMR studies of paramagnetic proteins such as Pdx is the broadening of resonances in the vicinity of the metal cluster due to paramagnetism. The extent to which a resonance is broadened by paramagnetic relaxation is proportional to the gyromagnetic ratio of the nucleus being observed. For this reason, 15N and 13C NMR spectroscopy are useful structural probes in the vicinity of the metal center of Pdx. Both 15N and 13C have lower gyromagnetic ratios and so are less affected by the paramagnetism of the metal center than 1H. Using a combination of residue-selective isotope labeling and selective decoupling NMR experiments, we have sequence-specifically identified almost all of the amide 15N and carbonyl 13C resonances in the metal binding site of Pdx for both oxidation states. (44,49) We are also developing 13C-detected 2D NMR methods that will remove the need for selective labeling in order to accomplish sequential NMR assignments near paramagnetic centers in proteins (61). In this effort we are being aided by 13C direct detection available with the cryoprobe on our Bruker 800 MHz NMR spectrometer (SCMF800).Enzymes in the methionine salvage pathway: structure and function
The methionine salvage cycle is a ubiquitous biochemical pathway the maintains methionine levels in vivo by recycling the thiomethyl moiety of methionine through a degradation pathway that leads from S-adenosyl methionine (SAM) through methylthioadenosine (MTA) (See Scheme 1). The ribose ring of MTA provides the carbon skeleton for acyclic intermediates in the salvage pathway. The structure and function of two enzymes in the pathway, the enolase-phosphatase E1 and acireductone dioxygenase (ARD) are under investigation in our laboratory. Both of these enzymes were discovered in the laboratory of Prof. R. Abeles. E1 and ARD catalyze subsequent steps in the methionine recycle pathway in Klebsiella pneumoniae.
Both enzymes are monomeric and relatively small in size. E1 is 24 kDa (229 residues) and ARD is 18 kDa (176 residues). The genes for both have been cloned into pET expression vectors, and high level expression of both enzymes has been achieved in E. coli. All of these factors make these enzymes, for which no crystal structures exist and which are without homologues in the PDB database, attractive targets for NMR structure/function studies.
A particularly interesting feature of ARD is that its functionality changes depending on which metal ion is bound to it. ARD to which Ni(II) (NiARD) is bound catalyzes the decomposition of acireductone substrate to the (n-2) carboxylic acid, carbon monoxide and formate. From the same substrate, ARD to which Fe(II) is bound generates the (n-1) a-keto acid and formate. The function (if any) of CO is unknown [4a, 5a, 6a]. However, CO has been implicated as a potential neurotransmitter in vertebrates [7a].
We have determined the solution structure of Ni-ARD [62,70)(See Figure 2), and have begun an investigation into the roles played by the metal ions in the course of the chemistry catalyzed by ARD [56]. Recently, we have also determined the structure of the Fe-containing form ARD (Figure 2), and observed an interesting structural entropy switch that is triggered by the metal ion bound in the active site, and results in extensive changes in secondary structural features of the protein.
Fig. 2. Comparison of the structures of ARD (A) and ARD' (B). Letters reference to the ARD sequence as follows: A (Ala 2-Phe 6), B (Leu 15-Ser 18), C (Glu 23-Lys 31), E (Thr 50-Tyr 57), E' (Ile 61-Lys 68), F (Ser 72-Leu 78), G (Lys 85-Glu 90), H (Phe 92-Glu 95), I (Arg 104-Val 107), J (Gly 111-Ile 117), K (Glu 120-Leu 125), L (Asn 129-Ile 132), M (His 140-Met 144), N (Phe 150-Phe 156), O (Gly 161-Gly 168), P (Ile 171-Ala 174). The positions of metal ions are indicated by blue (Ni+2) and gray (Fe+2) spheres. Residues 157-175 (loop O and helix P in ARD) are disordered in ARD', and so for clarity are not shown in the ARD' structure. We are using a wide variety of tools in this project besides structural NMR methods. In collaboration with Prof. Barry Snider, we developed a simple synthesis for substrate analogs of I, the precursor of acireductone II [66]. We are now in a position to examine the mechanism of this enzyme using enzymological techniques. Molecular biological methods are also being employed by our lab for this work. We are currently mutating residues the active site of ARD to observe the effect that these mutations have on expression and activity. We have also begun examining structure and function of the human homologue of ARD.
We are beginning the structural and functional characterization of the enolase-phosphatase E1, that catalyzes the conversion of the acyclic diketone intermediate I (Scheme I) to the enol phosphate, followed by dephosphorylation to yield aci-reductone II. We have expressed E1 in E. coli and have performed preliminary enzymatic activity assays and multidimensional NMR characterization of 15N,13C-labeled E1 [67].
Previous Projects
These are projects that we are not currently working on, but that are an important part of the group history, and we could probably be talked into working on again for the right person (or the right price).
Closed-shell ion pair structure and dynamics
Ion pairing is a phenomenon of considerable importance in chemical and biological systems, and has been studied by physical and spectroscopic methods for many years. NMR spectroscopy permits us to examine the time-average structures and dynamics of ion pairs. Of particular interest to us are the closed-shell ion pairs formed in non-polar solvents by large lipophilic cations such as quaternary ammonium ions and small hard anions such as chloride and tetrahydridoborate (BH4-). Such ion pairs are important in organic phase-transfer chemistry, and insight into their behavior will be used to design improved catalysts for phase-transfer reactions. Our observations of interionic NOEs in the tetrabutylammonium tetrahydridoborate (TBABH4) ion pair in CDCl3 showed that structural studies of ion pairs in non-polar media by NMR are feasible [19]. Temperature effects on the interionic NOE in TBABH4 and related ion pairs indicated that aggregation was probably taking place in these moderately concentrated solutions [24], and self-diffusion coefficients of tetrabutylammonium chloride relative to those measured for tetrabutylsilane in CDCl3 by pulsed field gradient NMR methods yielded an estimate of aggregate size as a function of temperature and concentration [34]. More recently, we have shown that the cation and anion diffuse at the same rate using borohydride anion diffusion as a probe [41]. The dynamics of the anion reorientation in the TBABH4 ion pair under a variety of conditions were characterized by 11B{1H}, 10B{1H} NOE and 11B, 10B relaxation measurements [29]. We are currently analyzing the results of 13C{1H} and 15N{1H} NOE and 13C,15N relaxation experiments in order to characterize the dynamics of the cation and overall aggregate motion in the TBABH4 ion pair [42]. Using selectively deuterated tetrabutylammonium salts, we are examining interionic NOEs in aggregates of tetrabutylammonium chloride in chloroform. Finally, we are continuing our investigation of complex ion pairs involved in asymmetric reductions of ketones in order to understand the mechanism of asymmetric induction. We will then apply this information to the design of more selective phase transfer catalysts (PTCs). The extension of NOE methodology to the study of more complex ion pairs with rotating-frame NOEs was accomplished using the ion pair derived from quinine by quaternarization with benzyl chloride ("benzylquininium chloride" or BQCl), followed by anion exchange with NaBH4 to yield "benzylquininium tetrahydridoborate" or BQBH4 [22]. This reagent has been shown to yield modest asymmetry in the reduction of prochiral ketones, and we have used the structural model to improve the asymmetric induction by rational catalyst redesign [51].
We have found further practical application of our model by using it to modify the reaction conditions under which asymmetric PTC reductions are performed. We have achieved an enantiomeric excess (ee) of 86% in the PTC reduction of a prochiral ketone that under standard PTC conditions yields only a 30% ee (Pochapsky and Hofstetter, patent pending). Finally, we have developed methods for examining ion pair structures using 19F{1H} NOEs [53].
Amino acid interaction free energies for protein folding simulations: experimental measurements and theoretical considerations
The protein folding problem remains one of the major unsolved challenges of structural biology. Although much has been learned experimentally concerning the later stages of folding, the initial collapse of the polypeptide chain to a compact state is still poorly understood. This is a critical step, since much of the search space is excluded at the point of collapse, and any algorithm which successfully folds a protein based on sequence information must be able to select those compact states which are most likely to form. The most important component of such a search is information concerning the interaction free energies of amino acid side chains, and for this, a set of pairwise interaction potentials is required. Many such potential tables have been devised, often from statistics concerning interaction frequencies from the protein structure data base [1a]. However, it has been shown recently that such potentials are likely to contain systematic errors in estimates of side chain interaction free energies, and as such may not be suitable for realistic folding simulations [2a].Our approach to this problem is two-fold. Experimentally, we are attempting to measure relative interaction free energies using affinity chromatography. We covalently bind functionality identical to a particular amino acid side chain to silica gel, and use this bonded phase as a stationary phase for liquid chromatography. Separation factors for amino acid derivatives in aqueous media on this support are then related to the relative free energies of interaction between the analytes and the bound side chain. To date, we have prepared phenylalanine and leucine side chain phases [26] . We have established that aromatic-aromatic and aromatic-aliphatic interactions are qualitatively different than aliphatic-aliphatic interactions, both in magnitude and in the nature of their temperature dependence. Our work suggests that aromatic amino acid side chains play a critical role in the initiation of the collapse of the polypeptide chain, and are likely to also be important in mediating protein-protein and protein-substrate interactions [46]. Besides experimental measurements, we also use lattice model Monte Carlo simulations to determine the minimal requirements of a potential for use in folding simulations. Our recent publications discuss the accuracy required for such a potential and test our theoretical predictions against a well-characterized lattice model for folding [36, 3a].
Publications
1. "Composition and Performance of Distillate Recycle Solvents from the SRC-I Process", (R. A. Winschel, T. C. Pochapsky and F. P. Burke) Fuel 60, 562-573 (1981).
2. "Chromatographic Separation of the Enantiomers of 2-Carboalkoxy-indolines and N-Aryl-a-Amino Esters on Chiral Stationary Phases Derived from N-(3,5-dinitrobenzoyl)-a-Amino Acids", (W. H. Pirkle, T. C. Pochapsky, G. S. Mahler and R. E. Field) J. Chromatogr. 348, 89-96 (1985).
3. "Preparation of N-(2-Naphthyl)-2-Amino Acids and Esters of High Enantiomeric Purity", (W. H. Pirkle and T. C. Pochapsky) J. Org. Chem. 51, 102-105 (1986).
4. "A New, Easily Accessible Reciprocal Chiral Stationary Phase for the Chromatographic Separation of Enantiomers", (W. H. Pirkle and T. C. Pochapsky) J. Am. Chem. Soc. 108, 352-354 (1986).
5. "Intermolecular 1H{1H} Nuclear Overhauser Effects in Diastereomeric Complexes: Support for a Chromatographically-Derived Chiral Recognition Model", (W. H. Pirkle and T. C. Pochapsky) J. Am. Chem. Soc. 108, 5627-5628 (1986).
6. "Useful and Easily Prepared Chiral Stationary Phases for the Direct Chromatographic Separation of the Enantiomers of a Variety of Derivatized Amines, Amino Acids, Alcohols and Related Compounds" (W. H. Pirkle, T. C. Pochapsky, G. S. Mahler, D. E. Corey, D. S. Reno, and D. M. Alessi) J. Org. Chem. 51, 4991-5000 (1986).
7. "Generation of Extreme Selectivity in Chiral Recognition", (W. H. Pirkle and T. C. Pochapsky) J. Chromatogr. 369, 175-177 (1986).
8. "Chiral Stationary Phases for the Direct Liquid Chromatographic Separation of Enantiomers" (W. H. Pirkle and T. C. Pochapsky) in Advances in Chromatography; Giddings, J. C.; Grushka, E.; Brown, P., Eds.; Marcel Dekker: New York, 1987; Vol. 27, pp. 73-127.
9. "Chiral Molecular Recognition in Small Bimolecular Systems: A Spectroscopic Investigation into the Nature of Diastereomeric Complexes", (W. H. Pirkle and T. C. Pochapsky) J. Am. Chem. Soc. 109, 5975-5982 (1987).
10. "Direct Chromatographic Separation of Diol Enantiomers", (W. H. Pirkle, T. C. Pochapsky G. S. Mahler, and M. H. Hyun) J. Chromatogr. 388, 307-314 (1987).
11. "Chiral Stationary Phases" (T. C. Pochapsky) Biochromatography 2, 28-36 (1987).
12. "Separation of Some Enantiomeric Di- and Tripeptides on Chiral Stationary Phases", (W. H. Pirkle, T. C. Pochapsky, D. M. Alessi and M. H. Hyun) J. Chromatogr., 398, 203-209 (1987).
13. "Separation of the Stereoisomers of a Homologous Series of bis-Amides on Chiral Stationary Phases", (W. H. Pirkle and T. C. Pochapsky) Chromatographia 25, 652-654 (1988).
14. "Probing the Mechanisms of Macromolecular Recognition: The Cytochrome b5-Cytochrome c Complex", (K. K. Rodgers, T. C. Pochapsky and S. G. Sligar) Science 240, 1657-1659 (1988).
15. "Consideration of Chiral Recognition Relevant to the Liquid Chromatographic Separation of Enantiomers", (W. H. Pirkle and T. C. Pochapsky) Chemical Reviews, 89, 347-362 (1989).
16. "Relationship Between Heme Binding Site Structure and Heme Orientations of Two Ferrocytochrome b5s. A Study in Prosthetic Group Recognition", (T. C. Pochapsky, G. N. La Mar, S. G. Sligar and S. McLachlan) J. Am. Chem. Soc. 112, 5258-5265 (1990).
17. "Systematic Studies of Chiral Recognition Mechanisms", (W. H. Pirkle, T. C. Pochapsky, J. A. Burke III, and K. C. Deming) in Chiral Separations, Stevenson, D. and Wilson, I. D., Eds., Plenum Press, New York, 1988.
18. "1H NMR Study of the Influence of Hydrophobic Contacts on Protein-Prosthetic Group Recognition in Bovine and Rat Ferricytochrome b5", (K. B. Lee, G. N. La Mar, S. G. Sligar, T. C. Pochapsky, L. A. Kehres, E. M. Fujinara and K. M. Smith) Biochemistry, 29, 9623-9631 (1990).
19. "A Study of Ion Pair Solution Structure Using Nuclear Overhauser Effects: Interionic 1H{1H} and 11B{1H} NOEs in the (n-Butyl)4N+,BH4- Ion Pair" (T. C. Pochapsky and P. M. Stone) J. Am. Chem. Soc. 112, 6714-15 (1990).Return to text
20. "1H NMR Study of the Role of Heme Carboxylate Side Chains in Modulating Heme Pocket Structure and the Mechanism of Reconstitution of Cytochrome b5", (K. B. Lee, G. N. La Mar, T. C. Pochapsky, R. K. Pandey, I. N. Rezzano, K. E. Mansfield and K. M. Smith) Biochemistry 30, 1878-1886 (1991).
21. "1H NMR Identification of a b-Sheet Structure and Description of Folding Topology in Putidaredoxin", (X. M. Ye and T. C. Pochapsky) Biochemistry 30, 3850-3856 (1991).
22. "Interionic Contacts in Complex Ion Pairs Detected by Rotating Frame Nuclear Overhauser Effects", (T. C. Pochapsky, P. M. Stone and S. S. Pochapsky) J. Am. Chem. Soc. 113, 1460-1462 (1991).Return to text
23. "Theory and Design of Chiral Stationary Phases for the Direct Chromatographic Separation of Enantiomers", (W. H. Pirkle and T. C. Pochapsky) J. Chromatogr. Sci. 47, 783-814 (1990).
24. "The Sign of the Nuclear Overhauser Effect as a Function of Temperature in Contact Ion Pairs", (T. C. Pochapsky, P. M. Stone and E. Callegari) J. Chem. Soc., Chem. Comm. 178-179 (1992).Return to text
25. "1H NMR Sequential Assignments and Identification of Secondary Structural Elements in Oxidized Putidaredoxin, an Electron Transfer Protein from Pseudomonas", (T. C. Pochapsky , X. M. Ye and S. S. Pochapsky) Biochemistry 31, 1961-1968 (1992).Return to text
26. "A Chromatographic Approach to the Determination of Relative Free Energies of Interaction Between Hydrophobic and Amphiphilic Amino Acid Side Chains", (T. C. Pochapsky and Q. Gopen) Protein Science 1, 786-795 (1992).Return to text
27. "Interpretation of Hyperfine Shift Patterns in Ferricytochromes b5 in Terms of Angular Position of the Heme: A Sensitive Probe for Peripheral Heme Protein Interactions", (K.-B. Lee, G. N. La Mar, T. C. Pochapsky, K. E. Mansfield, K. M. Smith and S. G. Sligar) Biochem. Biophys. Acta 1202, 189-199 (1993).
28. "Characterization of Hyperfine-Shifted 1H Resonances in Oxidized and Reduced Putidaredoxin", (T. C. Pochapsky and G. Ratnaswamy) Mag. Reson. Chem. 31, S73-S77 (1993).
29. "Closed-Shell Ion Pair Structure and Dynamics: Steady State 1H{1H}, 10B{1H} and 11B{1H} Nuclear Overhauser Effects and 10B, 11B Nuclear Relaxation of Tetraalkylammonium Tetrahydridoborates in Ion-Pairing and Dissociative Solvents", (T. C. Pochapsky , A.-P. Wang and P. M. Stone) J. Am. Chem. Soc. 115, 11084-11091 (1993).Return to text
30. "Backbone 1H and 15N Sequential Assignments in Carbonmonoxy Sperm Whale Myoglobin", (Y. Theiralt, C. Dalvit, T. C. Pochapsky, M. Chiu, P. E. Wright and S. G. Sligar), Journal of Biomolecular NMR 4, 491-504 (1994).
31. "An NMR-Derived Model for the Solution Structure of Oxidized Putidaredoxin", (T. C. Pochapsky, X. M. Ye, G. Ratnaswamy and T. A. Lyons), Biochemistry 33, 6424-6432 (1994).Return to text
32. "Redox-Dependent 1H NMR Spectral Features and Tertiary Structural Constraints on the C-Terminal Region of Putidaredoxin", (T. C. Pochapsky, G. Ratnaswamy and A. Patera), Biochemistry 33, 6433-6441 (1994).Return to text
33. "Conversion of a Fe2S2 Ferredoxin into Ga+3 Rubredoxin", (S. Kazanis, T. C. Pochapsky, T. M. Barnhart, J. E. Penner-Hahn, U. A. Mirza and B. T. Chait) J. Am. Chem. Soc. 117, 6625-6626 (1995).
34. "Closed-Shell Ion Pair Aggregation in Non-Polar Solvents Characterized By NMR Diffusion Measurements", (S. S. Pochapsky, T. C. Pochapsky and H.P. Mo) J. Chem. Soc., Chem. Comm. 2513 (1995).Return to text
35. "Redox-Dependent Dynamics of Putidaredoxin Characterized by Amide Proton Exchange", (T. A. Lyons, T. C. Pochapsky and G. Ratnaswamy), Protein Science 5, 627-639 (1996).Return to text
36. "Monte Carlo Simulations of Protein Folding Using Inexact Potentials: How Accurate Must Parameters Be In Order to Preserve Essential Features of the Energy Landscape?", (A. F. Pereira de Araujo and T. C. Pochapsky), Folding and Design 1, 299-314 (1996). Return to text
37. "A structure-based model for cytochrome P450cam-putidaredoxin interactions", (T. C. Pochapsky , T. A. Lyons, S. Kazanis, T. Arakaki and G. Ratnaswamy), Biochimie 78, 723-733 (1996).Return to text
38. "Intermolecular Interactions Characterized by Nuclear Overhauser Effects", (T. C. Pochapsky, H. Mo), Progress in Nuclear Magnetic Resonance Spectroscopy 30, 1-38 (1997).
39. "Estimates for the Potential Accuracy Required in Realistic Folding Simulations and Structure Recognition Experiments", (A. F. Pereira de Araujo and T. C. Pochapsky), Folding and Design 2, 135-139 (1997).
40. "Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Fe2S2 ferredoxin", (S. Kazanis and T. C. Pochapsky), J. Biomol. NMR 9, 337-346 (1997).
41. "Self-diffusion coefficients of paired ions", (H. Mo and T. C. Pochapsky), J. Phys. Chem. B. 101, 4855-4486 (1997).Return to text
42. "Closed-shell ion pairs: Cation and aggregate dynamics of tetraalkylammonium salt in an ion-pairing solvent" (H. Mo, T. C. Pochapsky , A. Wang and P. Stone Wilkinson), J. Am. Chem. Soc. 119, 11666-11673 (1997).Return to text
43. "The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S" (T. C. Pochapsky , M. Kuti and S. Kazanis) J. Biomol. NMR 12, 407-415 (1998).
44. "Redox dependence of hyperfine-shifted 13C and 15N resonances in putidaredoxin" (Nitin U. Jain and T. C. Pochapsky) J. Am. Chem. Soc 120, 12984-12985 (1998).Return to text
45. "Solution structure and dynamics of a serpin reactive site loop using interleukin 1b as a presentation scaffold" (T. C. Pochapsky, C. C. Arico-Muendel, A. Patera, M. Kuti and A. J. Wolfson) Protein Engineering 12, 189-202 (1999).
46. "Thermodynamics of interactions between amino acid side chains: Experimental differentiation of aromatic-aromatic, aromatic-aliphatic and aliphatic-aliphatic side chain interactions in water" (A. F. Pereira de Araujo, T. C. Pochapsky and B. Joughin) Biophys. J. 76, 2319-2328 (1999).Return to text
47. "A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas" (T. C. Pochapsky, H. Mo and S. S. Pochapsky), Biochemistry 38, 5666-5676 (1999).Return to text
48. "A refined model for the solution structure of oxidized putidaredoxin" (T. C. Pochapsky , N. U. Jain, M. Kuti, T. A. Lyons and J. Heymont) Biochemistry 38, 4681-4690 (1999).Return to text
49. "A new assignment strategy for the hyperfine shifted 13C and 15N resonances in Fe2S2 ferredoxins" (N. U. Jain and T. C. Pochapsky), Biochem. Biophys. Res. Commun. 258, 54-59 (1999).Return to text
50. "1H, 13C and 15N assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae" (H. Mo, Y. Dai, T. C. Pochapsky and S. S. Pochapsky) J. Biomol. NMR 14, 287-288 (1999).
51. "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?" (T. C. Pochapsky, C. Hofstetter and P. S. Wilkinson) J. Org. Chem. 64, 8794-8800 (1999).Return to text
52. "Designed molecular recognition: A commentary on possible design elements" (T. C. Pochapsky) Enantiomer 4, 437-444 (1999).
53. "BF4- as a probe for ion pair solution structure using interionic one- and two-dimensional 19F{1H} NOEs" (C. Hofstetter and T. C. Pochapsky) Magn. Reson. Chem. 38, 90-94 (2000).Return to text
54. "Site-selective deuterium labeling of the tetrabutylammonium cation" (M. J. Heinsen and T. C. Pochapsky) J. Labelled Compounds and Radiopharmaceuticals 43, 473-480 (2000).
55. "Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin" (T. C. Pochapsky , M. Kostic, N. Jain and R. Pejchal) Biochemistry 40, 5602-5614 (2001). Return to text
56. "Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae" (Y. Dai, T. C. Pochapsky and R. H. Abeles) Biochemistry 40, 6379-6387 (2001).Return to text
57. "Nuclear magnetic resonance as a tool in drug discovery, metabolism and disposition" (T. C. Pochapsky and S. S. Pochapsky) Curr. Top. Med. Chem. 1, 427-441 (2001).
58. "A Molecular Level Study of Complex Formation between Putidaredoxin and Cytochrome P450 by Scanning Tunneling Microscopy" (R. Mukhopadhyay, L. L. Wong, K. K. Lo, T. C. Pochapsky H. A. O. Hill) Physical Chemistry Chemical Physics 641-646 (2002).
59. "XAS Investigation of the Structure and Function of Ni in Acireductone Dioxygenase" (F. Al-Mjeni, T. C. Pochapsky, T. Ju, and M. J. Maroney) Biochemistry 41, 6761-6769 (2002).
60. "Comparison of functional domains in vertebrate-type ferredoxins" (M. Kostic, T. C. Pochapsky, S. S. Pochapsky, J. Obenauer, H. Mo, G. M. Pagani and R. Pejchal) Biochemistry 41, 5978-5989 (2002).
61. "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)" (M. Kostic, T. C. Pochapsky and S. S. Pochapsky), J. Am. Chem. Soc. 124, 9054-9055 (2002).Return to text
62. "Modeling and experiment yields the solution structure of acireductone dioxygenase from Klebsiella" (T. C. Pochapsky, S. S. Pochapsky, T. Ju, H. Mo, F. Al-Mjeni and M. J. Maroney) Nature Struct. Biol. 9, 966-972 (2002).Return to text
63. "Structure and dynamics of paramagnetic proteins by NMR" (M. Kostic and T. C. Pochapsky) "Paramagnetic resonance in metallobiomolecules". ACS Symposium Series, 858, pp. 214-226, Oxford University Press (2003) .
64. "A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450cam-putidaredoxin couple" (S. S. Pochapsky, T. C. Pochapsky and J. W. Wei), Biochemistry 42, 5649-5656 (2003).Return to text
65. "A Conserved histidine in vertebrate type ferredoxins is critical for redox-dependent dynamics" (M. Kostic, R. Bernhardt and T. C. Pochapsky), Biochemistry 42, 8171-8182 (2003).Return to text
66. "Analogs of 1-phospho-2,3-dioxo-5-methylthiopentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca." (Y. Zhang, M. Heinsen, M. Kostic, G. Pagani, L. Hedstrom, T. Riera, I. Perovic, B. B. Snider and T. C. Pochapsky), Bioorganic and Medicinal Chemistry 12, 3847-3855 (2004).Return to text
67. "1H, 13C and 15N chemical shift assignments of an enolase-phosphatase, E1, from Klebsiella oxytoca." (M. Kostic and T. C. Pochapsky), J. Biomol. NMR 30, 359-360 (2004).Return to text
68. "Detection of a high-barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin" (J. Y. Wei, T. C. Pochapsky and S. S. Pochapsky), J. Am. Chem. Soc., 127, 6974-6976 (2005).Return to text
69. "OsARD1 is an immediate-early ethylene response gene involved in recycling of the ethylene precursor S-adenosylmethionine" (M. Sauter, R. Lorbiecke, B. OuYang, T. C. Pochapsky and G. Rzewuski) The Plant Journal 44, 718-729 (2005).
70. "A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings" (T. C. Pochapsky, S. S. Pochapsky , T. Ju, C. Hoefler and J. Liang) J. Biomol. NMR 34, 117-127 (2006).Return to text
71. "Comparison of the complexes formed by cytochrome P450cam with cytochrome b5 and putidaredoxin, two effectors of camphor hydroxylase activity" (L. Rui, S. S. Pochapsky & T. C. Pochapsky) Biochemistry 45, 3887-3897 (2006).Return to text
72. "Nickel in acireductone dioxygenase" (T. C. Pochapsky, T. Ju, M. Dang, R. Beaulieu, and G.M. Pagani) in "Nickel and Its Surprising Impact in Nature", Vol. 2 of 'Metal Ions in Life Sciences'; A. Sigel, H. Sigel, R. K. O. Sigel, Eds.; John Wiley & Sons, Ltd., Chichester, UK, 2007, in press.
Books
NMR for Physical and Biological Scientists: a one-semester course. Thomas C. Pochapsky and Susan Sondej Pochapsky, accepted for publication by Taylor & Francis Publishing, scheduled for release September 2006.
Other References
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3a. A. F. Pereira de Araujo, Folding and Design 2, 135-139 (1997).Return to text
4a. J. W. Wray and R. H. Abeles, J. Biol. Chem. 268, 21466-21469 (1993).Return to text
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6a. Y. Dai, P. C. Wensink and R. H. Abeles, J. Biol. Chem. 274, 1193-1195 (1999).Return to text
7a. A. Verma, D. J. Hirsch, C. E. Glatt, G. V. Tonnett and S. H. Snyder, Science 259, 381-384 (1993).Return to text
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