Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro.

We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (L) chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative asembly patterns in experiments with H and L separated prior to recombination are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. However, when L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).     

Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin

The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].     

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.     

Crotonase-catalyzed beta-elimination is concerted: a double isotope effect study

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.     

New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability

Tris(2-carboxyethyl)phosphine (TCEP) is a widely used substitute for dithiothreitol (DTT) in the reduction of disulfide bonds in biochemical systems. Although TCEP has been recently shown to be a substrate of the flavin-dependent sulfhydryl oxidases, there is little quantitative information concerning the rate by which TCEP reduces other peptidic disulfide bonds. In this study, mono-, di-, and trimethyl ester analogues of TCEP were synthesized to evaluate the role of carboxylate anions in the reduction mechanism, and to expand the range of phosphine reductants. The effectiveness of all four phosphines relative to DTT has been determined using model disulfides, including a fluorescent disulfide-containing peptide (H(3)N(+)-VTWCGACKM-NH(2)), and with protein disulfide bonds in thioredoxin and sulfhydryl oxidase. Mono-, di-, and trimethyl esters exhibit phosphorus pK values of 6.8, 5.8, and 4.7, respectively, extending their reactivity with the model peptide to correspondingly lower pH values relative to that of TCEP (pK = 7.6). At pH 5.0, the order of reactivity is as follows: trimethyl- > dimethyl- > monomethyl- > TCEP > DTT; tmTCEP is 35-fold more reactive than TCEP, and DTT is essentially unreactive. Esterification also increases lipophilicity, allowing tmTCEP to penetrate phospholipid bilayers rapidly (>30-fold faster than DTT), whereas the parent TCEP is impermeant. Although more reactive than DTT toward small-molecule disulfides at pH 7.5, all phosphines are markedly less reactive toward protein disulfides at this pH. Molecular modeling suggests that the nucleophilic phosphorus of TCEP is more sterically crowded than the thiolate of DTT, contributing to the lower reactivity of the phosphine with protein disulfides. In sum, these data suggest that there is considerable scope for the synthesis of phosphine analogues tailored for specific applications in biological systems.     

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.     

Two-dimensional nMR study of bleomycin and its zinc(II) complex: reassignment of 13C resonances.

Two-dimensional NMR experiments--one bond 1H-13C correlation spectroscopy and heteronuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode--have been employed to unambiguously assign all of the 13C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous 1H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731-2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The 13C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388-396; Dabrowiak et al., (1978) Biochemistry 17, 4090-4096) but are in agreement with those reported by Akkerman et al. (1988) (Magn. Reson. Chem. 26, 793-802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous 13C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].     

Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburina, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alpha domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements. While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state. The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.     

Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions

The cytochrome a and a3 sites in uninhibited, detergent-solubilized cytochrome c oxidase have been studied under a wide range of conditions using thin-layer spectroelectrochemistry. The observed absorbance changes at the alpha and Soret absorbance maxima have been used together to estimate the extents of reduction of cytochromes a and a3, using the absorbance properties of these cytochromes deduced from previous measurements employing ligand inhibition of cytochrome a3. The resulting Nernst plots, combined with the results of parallel studies on the carbon monoxide-inhibited enzyme (Ellis, W. R., Jr., Wang, H., Blair, D. F., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 161-167; Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 167-171), indicate that the cytochrome a site participates in anticooperative thermodynamic interactions which involve all three of the other metal sites in the protein. Using an analysis which resolves the intrinsic thermodynamic properties of the cytochromes from the effects of the intersite interactions, the pH, temperature, and ionic strength dependences of the cytochrome reduction potentials have been measured. The standard entropy of reduction of cytochrome a in the native enzyme is large and negative, in agreement with measurements on the carbon monoxide-inhibited enzyme. The reduction potential of cytochrome a is only moderately (less than -30 mV/pH unit) dependent upon pH, which implies that its reduction is linked to the uptake, on the average, of only about 0.5 protons at pH 7.0, and significantly less at the higher pH values relevant to the mitochondrial matrix. The thermodynamic properties of cytochrome a3 were found to be different in the two enzyme batches studied: in one batch, the cytochrome a3 reduction potential decreased steeply (about -56 mV/pH unit) with increasing pH, indicating stoichiometric (1 H+/e-) coupling of protonation to reduction. In the other batch, the cytochrome a3 potential was insensitive to pH below pH 7.5 and decreased at higher pH values in a manner suggesting coupling to an ionizable group with pKa near 7.8. The temperature dependence of the cytochrome a3 reduction potential indicates that its standard entropy of reduction is more positive than that of myoglobin, another high-spin metalloprotein heme, and significantly more positive than that of cytochrome a.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lambda cro repressor complex with OR3 DNA: 15N NMR observations

15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.     

Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols

The kinetics of the disulfide-coupled unfolding-refolding transition of a mutant form of bovine pancreatic trypsin inhibitor (BPTI) lacking Cys-14 and -38 were measured and compared to previous results for the wild-type protein and other modified forms. The altered cysteines, which were changed to serine in the mutant protein, are normally paired in a disulfide in the native protein but from disulfides with Cys-5 in two-disulfide kinetic intermediates during folding. Although the mutant protein could fold efficiently, the kinetics of both folding and unfolding were altered, reflecting the roles of these cysteines in the two-disulfide intermediates with "wrong" disulfides. The intramolecular rate constant for the formation of the second disulfide of the native mutant protein was more than 10(3)-fold lower than that for the formation of a second disulfide during the refolding of the wild-type protein. The observed rate of unfolding of the mutant protein was also lower than that of the wild-type protein, demonstrating that the altered cysteines are involved in the intramolecular rearrangements that are the rate-determining step in the unfolding of the wild-type protein. These results confirm the previous conclusion [Creighton, T.E. (1977) J. Mol. Biol. 113, 275-293] that the energetically preferred pathway for folding and unfolding of BPTI includes intramolecular rearrangements of intermediates in which Cys-14 and -38 are paired in disulfides not present in the native protein. The present results are also consistent with other, less detailed, studies with similar mutants lacking Cys-14 and -38 [Marks, C.B., Naderi, H., Kosen, P.A., Kuntz, I.D., & Anderson, S. (1987) Science (Washington, D.C.) 235, 1370-1371].     

Identification of bull protamine disulfides

We have identified the disulfide cross-links in bull protamine by titrating intact bull sperm with dithiothreitol (DTT) and following the modification of each cysteine residue with tritiated iodoacetate. The derivatization of each cysteine was monitored by a combination of HPLC, peptide mapping, and protein sequencing. Analyses of total free sulfhydryls show that all seven of the bull protamine cysteines are cross-linked as disulfides in mature sperm. The first disulfide is reduced at a DTT:protamine cysteine (DTT:Cys) ratio of 0.3 and the last at a ratio of 2.0. Intra- and intermolecular disulfides were identified by correlating the reduction of specific disulfides with the dissociation of protamine from DNA in partially reduced sperm and sperm treated with N,N'-ethylenedimaleimide, a bifunctional disulfide cross-linking agent. Three intermolecular and two intramolecular disulfides were identified. The results of these experiments demonstrate that the amino- and carboxy-terminal ends of the bull protamine molecule are folded inward toward the center of the molecule and are locked in place, each by a single intramolecular disulfide bridge. Three intermolecular disulfides cross-link neighboring protamine molecules around the DNA helix in such a manner that the protamines cannot be dissociated from DNA without first reducing the interprotamine disulfides.     

Tertiary interactions between transmembrane segments 3 and 5 near the cytoplasmic side of rhodopsin.

Previous studies [Yu, H., Kono, M., and Oprian, D. D. (1999) Biochemistry 38, xxxx-xxxx] using split receptors and disulfide cross-linking have shown that native cysteines 140 and 222 on the cytoplasmic side of transmembrane segments (TM) 3 and 5 of rhodopsin, respectively, can cross-link to each other upon treatment with the oxidant Cu(phen)3(2+). In this paper we show that although the 140-222 cross-link does not affect the spectral properties of rhodopsin, it completely and reversibly inactivates the ability of the receptor to activate transducin. Following on this lead we further investigate the cytoplasmic region of TM3 and TM5 and identify three additional pairs of residues that when changed to Cys are capable of forming disulfide cross-links in the protein: 140/225, 136/222, and 136/225. These disulfides are able to form without addition of the Cu(phen)3(2+) oxidant. Similar to the 140-222 cross-link, none of the additional disulfides affect the spectral properties of rhodopsin. Also like the 140-222 bond, the 136-222 disulfide completely and reversibly inactivates the light-dependent activation of transducin by the receptor. In contrast, the 140-225 and 136-225 disulfides have no effect on the ability of rhodopsin to activate transducin. The pattern of cross-linking observed in Cys and disulfide scans of the protein is consistent with helical secondary structure in TM3 from 130 to 142 and in TM5 from 218 to 225.     

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions: full expression of the intrinsic isotope effect during the O-deethylation of 7-ethoxycoumarin by liver microsomes from 3-methylcholanthrene-induced hamsters

The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method [D. B. Northrop (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., eds.), pp. 122-152, University Park Press, Baltimore] for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.     

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.     

Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity

Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a recent report, human ALR is a disulfide-bridged dimer (linked via C15-C124) with two free cysteine residues (C74 and 85) per monomer. The C15-124 disulfides are not critical for dimer formation and have insignificant impact on the dithiothreitol (DTT) oxidase activity of ALR. Although the crystal structure of rat ALR shows a proximal disulfide (C62-C65) poised to interact with the FAD prosthetic group [Wu, C. K., Dailey, T. A., Dailey, H. A., Wang, B. C., and Rose, J. P. (2003) Protein Sci. 12, 1109-1118], only flavin reduction is evident during redox titrations of the enzyme. ALR forms large amounts of neutral semiquinone during aerobic turnover with DTT. This semiquinone arises, in part, by comproportionation between flavin centers within the dimer. Surprisingly, cytochrome c is about a 100-fold better electron acceptor for ALR than oxygen when DTT is the reducing substrate. These data suggest that this poorly understood flavoenzyme may not function as a sulfhydryl oxidase within the mitochondrial intermembrane space but may communicate with the respiratory chain via the mediation of cytochrome c.     

Two-Dimensional NMR Study of Bleomycin and Its Zinc(II) Complex: Reassignment of 13C Resonances

Abstract

Two-dimensional NMR experiments-one bond ¹H- ¹³C correlation spectroscopy and hetero- nuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode-have been employed to unambiguously assign all of the ¹³C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous ¹H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731–2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The ¹³C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388–396; Dabrowiak et al., (1978) Biochemistry 17, 4090–4096) but are in agreement with those reported by Akkerman et al.(1988) (Magn. Reson. Chem. 26, 793–802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous ¹³C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

Smooth muscle myosin light chain kinase. Amino acid sequence at the site phosphorylated by adenosine cyclic 3',5'-phosphate-dependent protein kinase whether or not calmodulin is bound

Turkey gizzard smooth muscle myosin light chain kinase is a calmodulin-dependent enzyme containing 2 serine residues that can be phosphorylated by cAMP-dependent protein kinase. One of these sites can be phosphorylated only when calmodulin is not bound to the enzyme; the amino acid sequence around this site has been reported recently (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464). Here we report the sequence around the site that is phosphorylated by cAMP-dependent protein kinase whether or not calmodulin is bound: Lys-Ala-Ser(P)-Gly-Ser-Ser-Pro-Thr-Ser-Pro-Ile-Asn-Ala-Asp-Lys-Val-Glu-A sn-Glu- . This sequence conforms to the previously defined criteria for substrates of cAMP-dependent protein kinase.