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J Rumbley

Publications and source records attributed to J Rumbley.

6 recordsLinked to original sources
PubMed2001

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.

Amino Acids↗
PubMed1995

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)

Computer Simulation↗
PubMed1994

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.

Affinity Labels↗
PubMed1992

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.

Amino Acid Sequence↗
PubMedDate not supplied

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.

Animals↗