Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

Ferricytochrome c. Refolding and the methionine 80-sulfur-iron linkage.

The refolding of urea-denatured horse heart ferricytochrome c in the presence of imidazole, 0.5 M, pH 7.0, has been examined using stopped-flow and equilibrium measurements at 407.5 nm. Thermodynamically, imidazole-cytochrome c folds and unfolds via a single transition with [urea]1/2 of 5.9 M. Kinetically, the refolding is a triphasic process: (i) a slow, urea-independent phase, time constant of 22 +/- 6 s, and an amplitude of 10-13%; (ii) an intermediate reaction, with a slightly positive urea-dependent rate constant, average time constant of 150 ms; and (iii) a fast phase with negative urea dependence of the rate constant from 4-6 M urea and positive dependence above the 6 M concentration, with the largest time constant, 25 +/- 6 ms, at 5.8 M urea, the midpoint of the transition. The amplitudes of the intermediate and the fast phases exhibit inverse dependence on the final urea concentrations, favoring the intermediate form at higher concentrations, while maintaining an almost constant sum of the two amplitudes throughout the range. The temperature dependence of the three apparent rate constants for the refolding from denatured base-line to midpoint of the transition, 9 to 6.03 M urea, yields linear Arrhenius plots with activation energies of 14, 19, and 23 +/- 3 kcal/mol for the slow, intermediate, and rapid reactions, respectively. These findings show that the slow reaction, time constant in decaseconds , does not require, directly or indirectly, the coordination of Met-80-S to heme iron. The formation of this linkage during the folding of the urea-denatured protein in the absence of extrinsic ligand, however, does alter the course of the refolding process. From a comparison of the proposed mechanisms and of the kinetic parameters for the folding of urea-denatured and of guanidine hydrochloride-denatured ferricytochrome c, it has been suggested that the two systems are distinct in detail, although both systems exhibit the slow, decasecond process.

Ascorbate reduction of horse heart cytochrome c. A zero-energy reduction reaction.

The ascorbate reduction of horse heart ferricytochrome c in 0.05 M phosphate + 0.25 M sodium sulfate, at pH 7.3, as a function of temperature, 12-36 degrees C, and at alkaline pH 8.4 using stopped flow technique has been examined. The data have been analyzed in terms of a two-step mechanism, binding followed by reduction (Myer, Y.P., Thallam, K.K., and Pande, A. (1980) J. Biol. Chem. 255, 9666-9673). At neutral pH and up to about 26 degrees C, the first order reduction constant is independent of temperature, i.e. with zero or near-zero activation energy. At higher temperatures, it becomes temperature-dependent, increasing with increasing temperature with an activation energy of about 35 kcal/mol. The stability of the cytochrome c-ascorbate complex is enhanced in the range 12-26 degrees C, with an enthalpy and an entropy change of about 3 kcal/mol and 32 e.u., respectively. Above 26 degrees C, the stability of the complex decreases. At pH 8.4, the reduction reaction is biphasic, and the behavior of the rapid, ascorbate-dependent component is consistent with the proposed two-step mechanism. A pH change of 1.1 units increases the first order reduction constant by a factor of 6, while the stability constant of the complex decreases to about one-fourth its value. The slow component at pH 8.4 is ascorbate-independent, with a rate constant of 0.043 +/- 0.006 s-1. The zero or near-zero activation energy for the reduction reaction below 26 degrees C and the development of temperature dependence at higher temperatures constitute the bases for concluding that the reduction reaction occurs via tunneling at temperatures below 26 degrees C. The observed reduction constant is consistent with tunneling from a distance of about 16 A, with an energy barrier of about 35 kcal/mol.

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochrome c in the presence of 0.2 M KCl, 0.1 M NaClO4, and 0.2 M KNO3, in the pH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990) J. Protein Chem, 9, 379-387), the change from neutral to low acidic pH's in the presence of salts is a three-step process: state IIIs----state IIIS,a----state IIS----state IS, with pKa's of 3.5 +/- 0.2, 2.2 +/- 0.2, and 1.1 +/- 0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutral pH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State IIIS,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states IIS and IS are also essentially folded forms, but with a smaller degree of protein secondary structure. State IIS has a high-spin hexacoordinated heme iron with a water molecular and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state IS has a high-spin pentacoordinated heme iron, which is about 0.49 A out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state IIIa----state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).

Methionine-oxidized horse heart cytochrome c. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage.

The ascorbate reduction of the CT-cytochromes--two chemically generated forms of horse heart cytochrome c, FIII and FII, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)--has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate + 0.17 M NaCl buffer, pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction and the stability of the ascorbate dianion-protein complex, the former is dependent on the integrity of the structural-conformational state of the molecule.

Horse heart ferricytochrome c: conformation and heme configuration of low ionic strength acidic forms.

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochrome c in the pH range 6-0.8 and at the lowest possible ionic strengths, in water, and at 30 degrees C are reported. The neutral pH form, state III, changes to the acidic pH form, state I, through a three-step process: state III in equilibrium with IIIa in equilibrium with state II in equilibrium with state I, with pKa's of 3.6 +/- 0.3, 2.7 +/- 0.2, and 1.2 +/- 0.2, depending on the monitoring probe, respectively. State IIIa ferricytochrome c is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochrome c is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin, and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 A.

Kinetics of the reduction of horse heart ferricytochrome c. Ascorbate reduction in the presence and absence of urea.

The reduction of horse heart cytochrome c with ascorbate in the absence of urea and in its presence, 0 to 8 M, pH 7.0, has been investigated using a stopped flow technique and the absorptivity at 550 nm as the monitoring probes, and by using the rate of oxidizability with molecular oxygen. Reduction is found to be consistent with a mechanism involving (i) a urea-dependent equilibrium step between an ascorbate-reducible and an irreducible form, with a [urea]1/2 of 7.5 M and a reversion rate constant of 0.05 +/- 0.02 s-1, (ii) the binding of ascorbate to cytochrome c, with a binding constant of 5.9 M-1 in the absence of urea which decreases to a value of 2.7 M-1 above 5.5 M urea, and (iii) a reduction step, with a urea-independent rate constant of 2.9 +/- 0.3 s-1. This scheme is interpreted in terms of an electron-transfer pathway involving neither the classical "adjacent" attack nor attack at the exposed heme edge, i.e. "remote" attack, but rather, through an alternate pathway involving binding at some site other than the heme crevice opening and a migration path of rather low electron-transfer efficiency. The urea-linked ascorbate reduction step is th X2 in equilibrium D step of the urea denaturation mechanism (Myer, Y. P., MacDonald L. H., Verma, B. C., and Pande, A. J. (1980) Biochemistry 19, 199-207), and the 9 M urea form, D, is the irreducible form. Form X2 and the other intermediate form, X1, are found to be reducible directly by ascorbate, and not through reversion to the native form of the protein. both the integrity of the heme crevice and the polypeptide-organized structures are of little importance as far as ascorbate reducibility is concerned, but the integrity of the structural and protein functional changes reflecting the X2 in equilibrium D step of the mechanism directly or indirectly determines the reducibility of the protein.

Conformational and functional studies of chemically modified cytochrome c: nitrated and iodinated cytochromes c.

The purification of iodinated (E. B. McGowan and E. Stellwagen (1970), Biochemistry 9, 3074) and of nitrated (M. Sokolovsky et al. (1970), Biochemistry 9, 5113) cytochromes c resulted in the recovery from the former preparation of diiododityrosyl-cytochrome c (DIDT-) with modification of Tyr-67 and Tyr-74, and, from the latter, a mononitromonotyrosyl-cytochrome c (MNMT-), with modification of Tyr-67, and mononitrodityrosyl-cytochrome c (MNDT-), with the added modification of Tyr-48. The three purified preparations were conformationally characterized using pH-spectroscopy, circular dichroism, thermal denaturation, reducibility with ascorbate, autoxidation with molecular oxygen, and binding with CO. These results are related to the two aspects of biological function, reducibility, measured by NADH-cytochrome c reductase, and oxidizability, with cytochrome c oxidase, as well as to structure-function relationships in the protein. MNMT-cytochrome c was found to be, structurally and conformationally, a single isomer, reducible with ascorbate, with a small, but definite affinity for both oxidation with molecular oxygen and binding of CO. Conformationally, in both valence states of the metal atom, it represents a molecular form with native-like conformation with small but definite perturbations in the immediate vicinity of the heme group, reflected by the destabilization of the Met-80-S-Fe linkage. MNMT-ferricytochrome c exhibits a pK of 6.2 for the transformation of the low-spin, native-like spectral form II containing the 695-nm band to form lacking lacking the 695-nm band. The isomerization at pK = 6.2, when analyzed in terms of the isomerization of the native protein with a pK of 9.2 and the nature of the group involved, indicates that Tyr-67 is not involved in the isomerization of the modified preparation, and possibly not in the native protein as well. In terms of biological function, the partial derangement of redecibility (24%) and the unaltered oxidizability point to the functional significance of Tyr-67, and provide another example of selectivity between the two aspects of physiological functional function, in agreement with the two-function, two-path operational model of the protein. The MNDT- and DIDT-ferricytochromes c exhibited physicochemical properties indicative of gross derangement of both the conformation of the protein as well as of the coordination configuration of the metal atom. The complete inability to accept an electron from NADH-cytochrome c reductase in both cases, and the retention of 50% of the oxidizability property of DIDT-cytochrome c, were interpreted to be the result of conformational derangement, rather than the added modification of Tyr-48 or of Tyr-74.

Kinetics of unfolding and folding of horse heart ferricytochrome c with urea.

Kinetic data for the reversible folding and unfolding by urea of horse heart ferricytochrome c, in 0.05 M phosphate + 0.25 M Na2SO4 buffer, pH 7.0, in the region of the main denaturation transition, 4-9 M, are reported. Stopped flow technique and absorptivity at 695, 528, and 361 nm as the monitoring probes were used. The decay profiles in the region of the transition 6-7.2 M urea are adequately described by a rate law with two exponential decay terms, but a rate law with only a single term is found to be applicable at the lower and higher limits of the transition. The apparent rate constant for the fast phase exhibits urea dependence with a minimum value at about 6.5 M urea, while the apparent rate constant of the slow phase is found to be independent of urea and has a value of 0.04 +/- 0.02 s-1. The assignment of the two apparent rate constants to the respective steps and the characterization of the processes involved were carried out through correlation of the kinetic data to the results from equilibrium studies for urea denaturation ( Myer, Y. P., MacDonald, L. H., Verma, B. C., and Pande, A. (1980) Biochemistry 19, 199-207). A mechanism X1 in equilibrium X2 in equilibrium D, where the first step is the urea-dependent unfolding and folding, and the second, an apparent urea-independent process involving possibly reorganization of the unfolded form X2, has been proposed to account for the above findings. The X2 in equilibrium D process is further considered in light of various possibilities: the incorrect folding of the unfolded form, the alteration of heme iron coordination, and the cis-trans isomerization of proline.

Stepwise modification of the electrostatic charge of cytochrome c. Effects on protein conformation and oxidation-reduction properties.

Horse heart cytochrome c was progressively maleylated, and fractions containing increasing numbers of modified lysines were obtained. The 695 nm band was present in derivatives containing up to 14 maleylated residues. Circular dichroic spectra showed minor changes beginning with 8 substituted lysines; in derivatives with 14 or more maleylated lysines, circular dichroism indicated total disruption of the native conformation. The ionic strength dependence of the measured oxidation reduction potentials and second order rate constants of reduction with ascorbate varied as expected from application of Debye-Huckel theory to the differently charged derivatives. The thermodynamic oxidation-reduction potentials decreased with the increase in the number of negatively charged groups, in a manner similar to that observed for simple iron complexes.

ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase.

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.

Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c.

N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.