An amino acid code for protein folding.

Direct structural information obtained for many proteins supports the following conclusions. The amino acid sequences of proteins can stabilize not only the final native state but also a small set of discrete partially folded native-like intermediates. Intermediates are formed in steps that use as units the cooperative secondary structural elements of the native protein. Earlier intermediates guide the addition of subsequent units in a process of sequential stabilization mediated by native-like tertiary interactions. The resulting stepwise self-assembly process automatically constructs a folding pathway, whether linear or branched. These conclusions are drawn mainly from hydrogen exchange-based methods, which can depict the structure of infinitesimally populated folding intermediates at equilibrium and kinetic intermediates with subsecond lifetimes. Other kinetic studies show that the polypeptide chain enters the folding pathway after an initial free-energy-uphill conformational search. The search culminates by finding a native-like topology that can support forward (native-like) folding in a free-energy-downhill manner. This condition automatically defines an initial transition state, the search for which sets the maximum possible (two-state) folding rate. It also extends the sequential stabilization strategy, which depends on a native-like context, to the first step in the folding process. Thus the native structure naturally generates its own folding pathway. The same amino acid code that translates into the final equilibrium native structure-by virtue of propensities, patterning, secondary structural cueing, and tertiary context-also produces its kinetic accessibility.

DNA hydrolysis by monoclonal autoantibody BV 04-01.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Signal transmission between subunits in the hemoglobin T-state.

To study allosteric mechanism in hemoglobin, a hydrogen-exchange method was used to measure ligand-dependent changes in structural free energy at defined allosterically sensitive positions. When the two alpha-subunits are CN-met liganded, effects can be measured locally, within the alpha-subunit, and also remotely, within the beta-subunit, even though the quaternary structure remains in the T conformation. When the two beta-subunits are liganded, effects occur at the same positions. The effects seen are the same, independently of whether ligands occupy the alpha-chain hemes or the beta-chain hemes. Control experiments rule out modes of energy transfer other than programmed cross-subunit interaction within the T-state. Cross-subunit transfer may depend on pulling the heme trigger (moving the heme iron into the heme plane) rather than on liganding alone.

One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase.

The cytochrome bo3 ubiquinol oxidase from Escherichia coli is a member of the heme-copper superfamily of proton-pumping respiratory oxidases. An improved preparative protocol was desired that would minimize the potential damage during protein isolation of labile mutants of the oxidase. Variants of the oxidase containing a histidine tag at the carboxy-terminus of either subunit I, II or III were constructed. The constructs with the histidine tag on either subunit I or II successfully allowed the enzyme to be isolated with high purity in one step using Ni2+ affinity chromatography. The enzyme with the histidine tag on subunit II is particularly useful insofar as the enzyme isolated in this manner has little, if any, heterogeneity resulting from the presence of heme O in the low spin heme-binding site, i.e., cytochrome oo3 is minimized. The enzyme can be prepared in virtually any quantity very rapidly and is suitable for biophysical characterization. Cytochrome bo3 was prepared in either Triton X-100, sucrose monolaurate, or dodecyl maltoside. The enzyme isolated in the presence of either sucrose monolaurate or dodecyl maltoside contains approximately one equivalent of associated ubiquinone, whereas this is absent when Triton X-100 is used. However, the UV/vis absorbance and steady-state kinetic properties of the enzyme are virtually identical regardless of which detergent is used. These data are consistent with previous reports that cytochrome bo3 contains an equivalent of 'tightly associated' ubiquinone, but clearly demonstrate that this quinone can be removed without damaging the enzyme and is not critical to the maintenance of the native structure of the oxidase.

Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo3 complex: implications for a Q(H2)-loop proton translocation mechanism.

The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H2)). One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H2) pool in the membrane. The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908-28912]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, these observations strongly suggest that there must be at least two UQ(H2) binding sites that are in rapid equilibrium with the UQ(H2) pool under turnover conditions. Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H2) binding sites were to function in a Q(H2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 131-136]. In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: origins of catalytic activity.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Reaction of cytochrome bo3 with oxygen: extra redox center(s) are present in the protein.

The reaction of oxygen with cytochrome bo3, a quinol oxidase from Escherichia coli, has been studied by resonance Raman scattering after initiation of the reaction by CO photolysis in a continuous flow apparatus and by directly mixing the enzyme with oxygen. The high-frequency region of the spectrum was monitored to determine the time evolution of the spin, oxidation, and coordination states of heme O and the oxidation state of heme B by using newly established marker lines for each heme. Three phases of the reaction were detected. In phase I, complete in 75 microseconds, O2 reacted with heme O and formed a low-spin ferric or ferryl adduct without significant oxidation of heme B. In phase II, between 75 and 120 microseconds, a small fraction of heme B was oxidized. In phase III, at approximately 1 s, the majority of heme B was oxidized and heme O reverted to a high-spin ferric state. The high rate of oxygen reduction at heme O to the three- or four-electron reduced level, despite a very low rate of heme B oxidation, indicates that there are electron donors active in the enzyme other than the metal centers. Assays of our enzyme preparations rule out a quinol in the tight binding (QH) site as a possible donor but instead suggest electron donation from the protein matrix, such as from tryptophans or tyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Interconversion of fast and slow forms of cytochrome bo from Escherichia coli.

The fully oxidized fast form of cytochrome bo from Escherichia coli is shown to convert spontaneously to a slow form when stored at -20 degrees C in 50 mM potassium borate, pH 8.5, containing 0.5 mM potassium EDTA. Evidence for the conversion, and that the form produced is analogous to the slow form of bovine heart cytochrome c oxidase, comes from (a) decreases in the extents of fast (k = 1-2 x 10(3) M-1 s-1) H2O2 binding and fast (k = 20-30 M-1 s-1) cyanide binding; (b) changes in the optical spectrum that are like those induced by formate, i.e., a blue shift in the Soret absorption band, loss of absorbance in the alpha and beta bands, and a red shift in the "630 nm" charge-transfer band; (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 8.6 and g = 3.71, and appearance of signals at g approximately 13, g = 3.14, and g = 2.58; and (d) appearance of a slow phase of reduction of heme o by dithionite. The mutant enzyme E286Q also converts to a slow form under the same conditions, as shown by (a) a decrease in the extent of fast H2O2 binding; (b) changes in the optical spectrum like those seen with wild-type enzyme; and (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 7.3 and g = 3.6 and appearance of signals at g approximately 13, g = 3.18, and g = 2.59.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the ubiquinol-binding site in the cytochrome bo3-ubiquinol oxidase of Escherichia coli.

The cytochrome bo3-ubiquinol oxidase, one of two ubiquinol oxidases in Escherichia coli, is a member of the heme-copper oxidase superfamily. The enzyme contains four protein subunits (I-IV) with apparent molecular masses of 58, 33, 22, and 17 kDa, respectively. Cytochrome bo3 catalyzes the 2-electron oxidation of ubiquinol and the reduction of molecular oxygen to water. Although the primary structures of all four subunits have been determined, the ubiquinol-binding site has not been investigated. The photoreactive radiolabeled azidoubiquinone derivative 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), which has been widely used in locating the ubiquinone-binding sites of other enzymes, was used to identify the subunit(s) involved in the binding of quinol to cytochrome bo3. When reduced by dithioerythritol, the azido-Q derivative functioned as a substrate with partial effectiveness, suggesting that azido-Q interacts with a legitimate quinol-binding site. When cytochrome bo3 was incubated with an 8-fold molar excess of azido-Q, illumination by UV light for 10 min resulted in a 50% loss of activity. The uptake of radiolabeled azido-Q by the oxidase complex upon illumination correlated with the photoinactivation. In the presence of the competitive inhibitor 2-heptyl-4-hydroxyquinoline or ubiquinol, the rate of azido-Q uptake and the loss of enzyme activity upon illumination decreased. Analysis of the distribution of radioactivity among the subunits after separation by SDS-polyacrylamide gel electrophoresis showed that subunit II was heavily labeled by azido-Q, but that the other subunits were not. This suggests that the ubiquinol-binding site of the cytochrome bo3 complex is located at least partially on subunit II.

Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli.

The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1. The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB. It appeared that rapid haem-haem electron transfer could still occur. The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.

Ligand-binding properties and heterogeneity of cytochrome bo from Escherichia coli.

Cyanide and formate induce spectral changes in E. coli cytochrome bo which are similar to those induced in bovine heart cytochrome-c oxidase (cytochrome aa3). Cyanide induces a red shift of 6 nm in the Soret band, whereas formate induces a blue shift of 2 nm. Cytochrome bo as purified shows multiphasic cyanide-binding kinetics. At least three phases can be seen with rate constants of 16, 1 and 0.1 M-1 s-1, respectively, at pH 7 and 20 degrees C. The enzyme after redox cycling ('pulsing') or in situ in E. coli membranes shows essentially monophasic binding with a rate constant of 15 M-1 s-1. Further evidence of heterogeneity in the enzyme as prepared comes from formate binding, which also shows at least three phases (rate constants of 1.4, 0.2 and 0.01 M-1 s-1, respectively, at pH 5 and 20 degrees C). The fast phase of cyanide binding is eliminated in less than 2 min by incubation with 40 mM formate, but the intermediate phase is unaffected by incubation for 3.5 h with 40 mM formate. Thus, the subpopulation that causes the fast phase of cyanide binding also causes the fast phase of formate binding. Formate-ligated cytochrome bo has similar cyanide-binding kinetics to the subpopulation that causes the slow phase of cyanide binding in cytochrome bo as prepared. It appears, from all this, that the subpopulations responsible for the fast and slow phase of cyanide binding are analogous to the 'fast' and 'slow' forms, respectively, of cytochrome aa3.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.