Biomedical subjects

G Ratnaswamy

Publications and source records attributed to G Ratnaswamy.

4 recordsLinked to original sources

PubMed1996

Redox-dependent dynamics of putidaredoxin characterized by amide proton exchange.

Multidimensional NMR methods were used to obtain 1H-15N correlations and 15N resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent 1H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 33:6433-6441). 15N assignments were made with NOESY-(1H/15N) HMQC and TOCSY-(1H/15N) HSQC spectra obtained using samples of Pdx uniformly labeled with 15N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYP101 upon the oxidation state of Pdx.

Bacterial Proteins↗

PubMed1996

A structure-based model for cytochrome P450cam-putidaredoxin interactions.

Putidaredoxin (Pdx) is a Fe2S2 ferredoxin which acts as the physiological reductant of cytochrome P-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined using NMR methods (Pochapsky et al (1994) Biochemistry 33, 6424-6432). 1H-15N correlations and redox-dependent amide exchange rates have also been described (Lyons et al (1996) Protein Sci 5, 627-639). Data obtained from mutagenesis and kinetic measurements concerning the interactions of Pdx and CYP101 are summarized. A model for the structure of the homologous ferredoxin adrenodoxin (Adx) is also described, and data concerning Adx activity are discussed in relation to this structure. The structures of Pdx and CYP101 were used as starting points for molecular modeling and molecular dynamics simulations. Close approach between the metal centers of the two proteins and interaction between aromatic residues on the surfaces of the proteins are premised. The resulting complex exhibits three intermolecular salt bridges, five intermolecular hydrogen bonds and a 12 A distance between the metal centers. The first direct observations of interaction between Pdx and CYP101 (by two-dimensional NMR of 15N-labeled Pdx in solution with CYP101) are described. The results of the NMR experiments indicate that conformational gating of the electron transfer complex between CYP101 and Pdx may be important.

Adrenodoxin↗

PubMed1994

An NMR-derived model for the solution structure of oxidized putidaredoxin, a 2-Fe, 2-S ferredoxin from Pseudomonas.

A model for the solution structure of oxidized putidaredoxin (Pdx), a 106-residue globular protein containing a Fe2S2 cluster, has been determined using homonuclear NMR methods. Pdx is the first of the class of Fe2S2Cys4 ferredoxins which act as electron-transfer partners for P-450 monooxygenases to be structurally characterized, and no crystal structure has been determined for Pdx or for any closely homologous protein. Pdx is the physiological redox partner of cytochrome P-450cam. A total of 878 NOE distance constraints, 66 phi angular constraints derived from NH-C alpha H coupling constants, and five paramagnetic broadening constraints were used in simulated annealing structural refinements to obtain a family of structures with pairwise rms deviations of 1.14 A for backbone atoms and 1.80 A for all non-hydrogen atoms. Paramagnetic broadening of resonances within a ca. 8-A radius of the metal cluster prevents the use of NMR-derived constraints in this region of the protein; structural constraints used to model the environment of the metal cluster were obtained from site-directed mutagenesis and model compounds and by comparison with known ferredoxin structures. Pdx retains a similar folding topology to other structurally characterized Fe2S2Cys4 ferredoxins but differs from the other ferredoxins in containing a significantly more compact structure in the C-terminal half of the protein.

Amino Acid Sequence↗

PubMed1994

Redox-dependent 1H NMR spectral features and tertiary structural constraints on the C-terminal region of putidaredoxin.

Putidaredoxin (Pdx) is a 106-residue Fe2S2 ferredoxin which acts as the physiological reductant and effector of cytochrome P-450cam. Pdx has two accessible oxidation states, Fe+3-Fe+3 (oxidized) and Fe+3-Fe+2 (reduced), and exhibits redox-dependent binding affinities for cytochrome P-450cam, with reduced Pdx binding over 100-fold more tightly than oxidized Pdx to the oxidized cytochrome P-450cam [Hintz, M. J., Mock, D. M., Peterson, L. L., Tuttle, K., & Peterson, J. A. (1982) J. Biol. Chem. 257, 14324-14332]. The analysis of two-dimensional 1H NMR experiments has yielded sequential 1H resonance assignments for the diamagnetic regions of the reduced form of Pdx, which are compared to those of oxidized Pdx, described previously [Ye, X. M., Pochapsky, T. C., & Pochapsky, S. S. (1992) Biochemistry 31, 1961-1968]. Increased unpaired electron-spin density on the metal cluster in reduced relative to oxidized Pdx increases the number of 1H resonances which are broadened by the metal cluster, and the pattern of paramagnetic broadening provides information concerning the placement of the metal cluster within the protein. Two-dimensional exchange experiments on half-reduced samples of Pdx indicate that electron self-exchange is slow on the chemical shift time scale, with a second-order rate constant < or = 66 M-1 s-1 at 290 K. Spectral changes unrelated to increases in unpaired electron-spin density are also observed. The largest changes of this type are observed for features structurally contiguous with the C-terminal region Pro 102-Trp 106. The C-terminal residue Trp 106 has been implicated in binding to cytochrome P-450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗