Plant coenzyme A biosynthesis: characterization of two pantothenate kinases from Arabidopsis.

In bacterial and animal coenzyme A (CoA) biosynthesis, pantothenate kinase (PANK) activity is critical in regulating intracellular CoA levels. Less is known about the role of PANK in plants, although a single plant isozyme from Arabidopsis, AtPANK1, was previously cloned and analyzed in vitro. We report here the characterization of a second pantothenate kinase of Arabidopsis, AtPANK2, as well as characterization of the physiological roles of both plant enzymes. The activity of the second pantothenate kinase, AtPANK2, was confirmed by its ability to complement the temperature-sensitive mutation of the bacterial pantothenate kinase in E. coli strain ts9. Knock-out mutation of either AtPANK1 or AtPANK2 did not inhibit plant growth, whereas pank1-1/pank2-1 double knockout mutations were embryo lethal. The phenotypes of the mutant plants demonstrated that only one of the AtPANK enzymes is necessary and sufficient for producing adequate CoA levels, and that no other enzyme can compensate for the loss of both isoforms. Real-time PCR measurements of AtPANK1 and AtPANK2 transcripts indicated that both enzymes are expressed with similar patterns in all tissues examined, further suggesting that AtPANK1 and AtPANK2 have complementary roles. The two enzymes have homologous pantothenate kinase domains, but AtPANK2 also carries a large C-terminal protein domain. Sequence comparisons indicate that this type of "bifunctional" pantothenate kinase is conserved in other higher eukaryotes as well. Although the function of the C-terminal domain is not known, homology structure modeling suggests it contains a highly conserved cluster of charged residues that likely constitute a metal-binding site.

Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins.

The oxidative folding of proteins consists of conformational folding and disulfide-bond reactions. These two processes are coupled significantly in folding-coupled regeneration steps, in which a single chemical reaction (the "forward" reaction) converts a conformationally unstable precursor species into a conformationally stable, disulfide-protected successor species. Two limiting-case mechanisms for folding-coupled regeneration steps are described. In the folded-precursor mechanism, the precursor species is preferentially folded at the moment of the forward reaction. The (transient) native structure increases the effective concentrations of the reactive thiol and disulfide groups, thus favoring the forward reaction. By contrast, in the quasi-stochastic mechanism, the forward reaction occurs quasi-stochastically in an unfolded precursor; i.e., reactive groups encounter each other with a probability determined primarily by loop entropy, albeit modified by conformational biases in the unfolded state. The resulting successor species is initially unfolded, and its folding competes with backward chemical reactions to the unfolded precursors. The folded-precursor and quasi-stochastic mechanisms may be distinguished experimentally by the dependence of their kinetics on factors affecting the rates of thiol--disulfide exchange and conformational (un)folding. Experimental data and structural and biochemical arguments suggest that the quasi-stochastic mechanism is more plausible than the folded-precursor mechanism for most proteins.

A role for intermolecular disulfide bonds in prion diseases?

The key event in prion diseases seems to be the conversion of the prion protein PrP from its normal cellular isoform (PrP(C)) to an aberrant "scrapie" isoform (PrP(Sc)). Earlier studies have detected no covalent modification in the scrapie isoform and have concluded that the PrP(C) --> PrP(Sc) conversion is a purely conformational transition involving no chemical reactions. However, a reexamination of the available biochemical data suggests that the PrP(C) --> PrP(Sc) conversion also involves a covalent reaction of the (sole) intramolecular disulfide bond of PrP(C). Specifically, the data are consistent with the hypothesis that infectious prions are composed of PrP(Sc) polymers linked by intermolecular disulfide bonds. Thus, the PrP(C) --> PrP(Sc) conversion may involve not only a conformational transition but also a thiol/disulfide exchange reaction between the terminal thiolate of such a PrP(Sc) polymer and the disulfide bond of a PrP(C) monomer. This hypothesis seems to account for several unusual features of prion diseases.

Structural determinants of oxidative folding in proteins.

A method for determining the kinetic fate of structured disulfide species (i.e., whether they are preferentially oxidized or reshuffle back to an unstructured disulfide species) is introduced. The method relies on the sensitivity of unstructured disulfide species to low concentrations of reducing agents. Because a structured des species that preferentially reshuffles generally first rearranges to an unstructured species, a small concentration of reduced DTT (e.g., 260 microM) suffices to distinguish on-pathway intermediates from dead-end species. We apply this method to the oxidative folding of bovine pancreatic ribonuclease A (RNase A) and show that des[40-95] and des[65-72] are productive intermediates, whereas des[26-84] and des[58-110] are metastable dead-end species that preferentially reshuffle. The key factor in determining the kinetic fate of these des species is the relative accessibility of both their thiol groups and disulfide bonds. Productive intermediates tend to be disulfide-secure, meaning that their structural fluctuations preferentially expose their thiol groups, while keeping their disulfide bonds buried. By contrast, dead-end species tend to be disulfide-insecure, in that their structural fluctuations expose their disulfide bonds in concert with their thiol groups. This distinction leads to four generic types of oxidative folding pathways. We combine these results with those of earlier studies to suggest a general three-stage model of oxidative folding of RNase A and other single-domain proteins with multiple disulfide bonds.

Recent improvements in prediction of protein structure by global optimization of a potential energy function.

Recent improvements of a hierarchical ab initio or de novo approach for predicting both alpha and beta structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic alpha-helical protein of 70 residues was predicted to within 4.3 A alpha-carbon (C(alpha)) rms deviation (rmsd) whereas, for other alpha-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 A C(alpha) rmsd. Whereas beta structures can now be predicted with the new procedure, the success rate for alpha/beta- and beta-proteins is lower than that for alpha-proteins at present. For the beta portions of alpha/beta structures, the C(alpha) rmsd's are less than 6.0 A for contiguous fragments of 30-40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a C(alpha) rmsd less than 6.0 A. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

Oxidative folding of proteins.

The oxidative folding of proteins is reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). The mutual effects of conformational folding and disulfide bond regeneration are emphasized, particularly the "locking in" of native disulfide bonds by stable tertiary structure in disulfide intermediates. Two types of structured metastable disulfide species are discerned, depending on the relative protection of their disulfide bonds and thiol groups. Four generic pathways for oxidative folding are identified and characterized.

Disulfide bonds and protein folding.

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.

Acceleration of oxidative folding of bovine pancreatic ribonuclease A by anion-induced stabilization and formation of structured native-like intermediates.

Phosphate anions accelerate the oxidative folding of reduced bovine pancreatic ribonuclease A with dithiothreitol at several temperatures and ionic strengths. The addition of 400 mM phosphate at pH 8.1 increased the regeneration rate of native protein 2.5-fold at 15 degrees C, 3.5-fold at 25 degrees C, and 20-fold at 37 degrees C, compared to the rate in the absence of phosphate. In addition, the effects of other ions on the oxidative folding of RNase A were examined. Fluoride was found to accelerate the formation of native protein under the same oxidizing conditions. In contrast, cations of high charge density or ions with low charge density appear to have an opposite effect on the folding of RNase A. The catalysis of oxidative folding results largely from an anion-dependent stabilization and formation of tertiary structure in productive disulfide intermediates (des-species). Phosphate and fluoride also accelerate the initial equilibration of unstructured disulfide ensembles, presumably due to non-specific electrostatic and hydrogen bonding effects on the protein and solvent.

Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A.

Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.

Conformational unfolding studies of three-disulfide mutants of bovine pancreatic ribonuclease A and the coupling of proline isomerization to disulfide redox reactions.

The equilibrium stability and conformational unfolding kinetics of the [C40A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNase A) have been studied. These mutants are analogues of two nativelike intermediates, des[40-95] and des[65-72], whose formation is rate-limiting for oxidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon addition of guanidine hydrochloride, both mutants exhibit a fast conformational unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isomerizations of Pro93 and Pro114. The amplitudes of the slow phase indicate that the two prolines, Pro93 and Pro114, are fully cis in the folded state of the mutants and furthermore that the 40-95 disulfide bond is not responsible for the quenching of Tyr92 fluorescence observed in the slow unfolding phase, contrary to an earlier proposal [Rehage, A., and Schmid, F. X. (1982) Biochemistry 21, 1499-1505]. The ratio of the kinetic unfolding m value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessible area, as in the wild-type protein, indicating that the unfolding pathway is not dramatically altered by the reduction of the 40-95 or 65-72 disulfide bond. The stabilities of the folded mutants are compared to that of wild-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, whereas that of des[65-72] is rate-limited by local conformational unfolding. The isomerization of Pro93 may be rate-limiting for the reduction of the 40-95 disulfide bond in the native protein and in the des[65-72] intermediate.

Proline isomerization in bovine pancreatic ribonuclease A. 1. Unfolding conditions.

The slow fluorescence unfolding phase of bovine pancreatic ribonuclease A is studied by stopped-flow kinetics and site-directed mutagenesis of tyrosines to phenylalanine and prolines to alanine. It is shown conclusively that this phase arises from two specific sources: Tyr92 reporting on the cis-trans isomerization of Pro93 and Tyr115 reporting on the cis-trans isomerization of Pro114. Previous studies have conjectured that the slow unfolding phase arises from only one source (Tyr92-Pro93 cis-trans isomerization) based primarily on studies of the homologous protein guinea pig ribonuclease A [Schmid, F. X., Grafl, R., Wrba, A., and Beintema, J. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872-876]; it is proposed here that Lys113 in the latter protein interferes with the isomerization of the Lys113-Pro114 peptide group. The site-directed mutations studied here enable the individual isomerizations of Pro93 and Pro114 to be monitored, providing an optical technique by which these well-defined molecular folding events can be studied, under both folding and unfolding conditions, and compared to molecular simulations. The time constants for Pro93 and Pro114 isomerization agree closely with those of our box model of proline isomerization under unfolding conditions, which had been derived from exhaustive statistical modeling of double-jump refolding data [Juminaga, D., Wedemeyer, W. J., Garduño-Júarez, R., McDonald, M. A., and Scheraga, H. A. (1997) Biochemistry 36, 10131-10145].

Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: a study of tyrosine-to-phenylalanine mutants.

Three tyrosine-to-phenylalanine mutants of ribonuclease A (Y25F, Y92F, and Y97F) are investigated for their enzymatic activities, molecular stabilities, and unfolding/refolding kinetics. These mutants exhibit 80, 90, and 80%, respectively, of the catalytic activity of the wild-type enzyme. Thermal, Gdn.HCl, and pH transition measurements indicate that Y25F and Y97F are less stable than the wild-type protein, whereas the bulk of the thermodynamic and kinetic evidence indicates that Y92F is as stable as the wild-type protein. Differences in molar extinction coefficients indicate that tyrosines 25, 92, and 97 contribute 38, 13, and 39%, respectively, to the absorption difference between the folded and unfolded states, in general agreement with previous studies but possibly indicating the contribution of a fourth tyrosine residue to account for the remaining 10%. Stopped-flow single- and double-jump kinetic experiments were carried out on the wild-type and three mutant proteins. At least one tyrosine residue besides tyrosine 92 contributes to the slow fluorescence-unfolding phase; the likely candidate for this residue is tyrosine 115 which monitors the cis-trans isomerization of the X-Pro114 peptide bond. Tyrosines 25 and 97 are involved in interactions that retard conformational unfolding and accelerate conformational refolding as well as the cis-trans proline isomerization of the slow-refolding phases, presumably by stabilizing the major beta-hairpin structure of RNase A. These interactions may contribute to the strong pH dependence of the folding and unfolding of ribonuclease A. In contrast, tyrosine 92 does not affect the folding and unfolding rates significantly. An improved "box" model of proline isomerization under unfolding conditions was derived from exhaustive fitting of all possible box models. The kinetic data support the identification of Pro93 as the proline whose isomerization distinguishes the slow-refolding species (USII and USI) from the other, faster-refolding species (Uvf, Uf, and Um), implying that Pro93 isomerizes in the slow-refolding reactions USI --> N and IN --> N. Similarly, Pro114 seems to distinguish between the very fast-refolding species Uvf and the fast-refolding species Uf. Lastly, Pro117 seems to distinguish the major slow-refolding species USII from the minor slow-refolding species USI and the medium-refolding species Um from the fast- and very fast-refolding species.

Kinetics of competitive binding with application to thrombin complexes.

The kinetics of competitive binding is treated analytically, allowing the rate constants to be determined accurately from simple experiments. The method is especially suited to situations where traditional approximations and numerical integration fail, e.g., when the dissociation constants are small or when the concentration of one receptor cannot be measured accurately. The method is applied to the competitive binding of hirudin to thrombin and anhydrothrombin and found to be accurate to a few parts in ten million. The fitted rate constants show that anhydrothrombin binds hirudin more weakly than thrombin, with a 2.6-fold increase in its dissociation constant. The small relative difference in binding free energy (0.6 kcal/mol indicates that anhydrothrombin is structurally similar to thrombin.

Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.

Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.