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P M Horowitz

Publications and source records attributed to P M Horowitz.

At least 55 records · Page 3Linked to original sources
PubMed1995

Photoincorporation of 4,4'-bis(1-anilino-8-naphthalenesulfonic acid) into the apical domain of GroEL: specific information from a nonspecific probe.

The use of noncovalent hydrophobic probes such as bis-ANS has become increasingly popular in gaining structural information about protein structure and conformation. While these probes have provided rich information about protein conformation, specific information has been limited. In this report, we extend the usefulness of the probe bis-ANS by showing that it can be covalently photoincorporated into various proteins. Using the chaperonin GroEL, we have shown that it is possible to locate important hydrophobic surfaces through photoincorporation and peptide sequencing. It has been proposed that hydrophobic surfaces on the chaperonin may be responsible for the binding of unfolded polypeptides. We show here that photoincorporation of bis-ANS is able to locate a distinct hydrophobic surface on GroEL. Incorporation of the bis-ANS occurs within a 45 residue fragment of the monomer near the middle of the primary sequence. Interestingly, photoincorporation occurs within this fragment in both tetradecamers and assembly-competent monomers. From the three-dimensional structure of GroEL, this region maps to the apical domain (residues 191-376), which has been implicated in polypeptide binding [Fenton, W. A., Kashi, Y., Furtak, K., & Horwich, A. L. (1994) Nature 371, 614-619]. In addition, the fluorescent properties of the probe are retained including the excitation and emission maxima and the sensitivity to the polarity of its environment. These results suggest that photoincorporated bis-ANS may be a useful probe for protein structure and dynamics.

Amino Acid Sequence↗
PubMed1995

The molecular chaperonin cpn60 displays local flexibility that is reduced after binding with an unfolded protein.

Steady-state fluorescence polarization was used to examine the chaperonin cpn60 that was covalently labeled with pyrene. Two compounds, 1-pyrenesulfonyl chloride or N-(1-pyrene)maleimide, were used to incorporate up to 8 mol of pyrene per mol of cpn60 14-mer. The fluorescence lifetime of the cpn60-pyrenesulfonyl chloride conjugate exhibited a double exponential decay: 5.36 ns, with a fractional contribution to the intensity of 7%, and 48.77 ns, with a fractional contribution to the intensity of 93%. These yield a second-order average lifetime of 45.58 ns at 20 degrees C. Analysis of the fluorescence polarization data for the pyrene probe by the Perrin-Weber treatment revealed the existence of two components that account for the depolarization. The fast component accounted for 24% of the depolarization at 20 degrees C. The rotational relaxation time for the cpn60 14-mer derived from the low viscosity part of the Perrin-Weber plot which accentuates the slow motion gave rho h = 1113 +/- 55 ns. When this value of rho h is compared with the rho h calculated based on the Stokes radius of cpn60 from ultracentrifugation, rho Stokes, it leads to rho h/rho Stokes = 0.4 which is considerably smaller than the value expected (rho h/rho Stokes = 1) or actually found in the cpn60-rhodanese complex (rho h/rho Stokes = 0.93). These considerations and the observed presence of the fast motion suggest that cpn60 is not a rigid protein. Analysis of the polarization data as a function of temperature, which is weighted more toward the fast motion, showed that the rotational relaxation time assessed by temperature variation is greatly increased (from 552.5 to 2591 ns) for the complex of cpn60 with partially folded rhodanese (34-kDa monomeric protein). No change in rho h was observed upon formation of the cpn60.ATP complex (rho h = 556.9 ns). These data indicate that there is local motion in the cpn60 14-mer molecule that can be frozen by formation of a binary complex with partially folded proteins. This conclusion is in keeping with results showing that the structure of cpn60 is generally stabilized when it forms complexes with passenger proteins (Mendoza, J. A., and Horowitz, P. M. (1994) J. Biol. Chem. 269, 25963-25965).

Chaperonin 60↗
PubMed1995

The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.

Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.

Escherichia coli Proteins↗
PubMed1995

Exposure of hydrophobic surfaces on the chaperonin GroEL oligomer by protonation or modification of His-401.

Hydrophobic exposure on the chaperonin GroEL is increased 6-10-fold after the protein is treated with the His-reactive reagent diethyl pyrocarbonate (DEP), or the solution pH is lowered to 5.5. The induced hydrophobic surfaces have the same 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding characteristics as unperturbed GroEL: a Kd approximately equal to 3.5 microM, a maximum intensity at approximately 500 nm, and an average fluorescence lifetime of approximately 8.0 ns. The pKa for the pH-induced transition is 6.6, most likely attributable to the only histidine in GroEL, His-401, located in the intermediate domain. The modification of one histidine residue per monomer upon DEP treatment is supported by the correlation between the change in the absorbance at 242 nm for the N-carbethoxyhistidyl derivative and the increase in bis-ANS fluorescence. GroEL at pH 5.5 is tetradecameric and can capture urea-denatured rhodanese and release it as active enzyme. The GroEL-rhodanese and release it as active enzyme. The GroEL-rhodanese complex is more stable to dissociation by 2.25 M urea than the complex formed at pH 7.8. We propose that His-401 is in a conformationally sensitive region such that protonation or modification can lead to increased exposure of hydrophobic surfaces capable of binding folding intermediates.

Anilino Naphthalenesulfonates↗
PubMed1995

Tetradecameric chaperonin 60 can be assembled in vitro from monomers in a process that is ATP independent.

The present work shows that monomers of cpn60 (groEL) formed at 2.5 M urea could be assembled to tetradecamers in a process that was independent of ATP. Reassembled cpn60 was able to assist the folding of urea unfolded rhodanese. When cpn60 was incubated at urea concentrations higher than 2.75 M, assembly of tetradecameric cpn60 did not occur after dialysis, and the presence of ATP did not stimulate the assembly process. The cpn60 used here did not display the previously reported ATP-dependent self-assembly of cpn60 monomers that required a higher urea concentration (4 M) for formation (Lissen et al. (1990) Nature 348, 339-342). Assembly and disassembly of cpn60 tetradecamers were followed as a function of the urea concentration by ultracentrifugation and gel electrophoresis in the presence of urea. The electrophoresis results demonstrate that there is rapid assembly of tetradecamers following preincubation and rapid removal of urea at concentrations lower than 2.5 M. Thus, previous methods monitored irreversible dissociation of cpn60, and the present results indicate that the cpn60 assembly requirements for ATP are dependent on pretreatment conditions.

Adenosine Triphosphate↗
PubMed1995

High hydrostatic pressure induces the dissociation of cpn60 tetradecamers and reveals a plasticity of the monomers.

Hydrostatic pressures up to 2 kbar have been used to form monomers from the 14-subunit oligomer of the chaperonin, Cpn60. The fluorescence of 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS), followed at high pressure, demonstrated an increase in hydrophobic exposure on dissociation. Cpn60 dissociated with first order kinetics. The transition occurred between 1.3 and 2 kbar (P50 = 1.75 kbar), and it was facilitated by MgATP (P50 = 1.1 kbar). With MgATP, the fluorescence showed a rapid first order phase (t1/2 = 3.7 min) in addition to a phase that was similar to the single phase for Cpn60 alone (t1/2 = 11.4 min). The bisANS fluorescence decreased slowly after depressurization, and the relaxation was faster at 25 degrees C (t1/2 = 58 h) than at 4 degrees C (t1/2 = 86 h) and faster still if the sample at 4 degrees C contained MgATP when it was pressurized (t1/2 = 18 h). There was no significant effect if the MgATP was added after depressurization. Analytical ultracentrifugation, after depressurization, confirmed that metastable monomers were produced that slowly reassociated to form the oligomers (t1/2 = 150 h at 25 degrees C). Immediately after depressurization, the monomers (a) had all three sulfhydryl groups exposed for labeling with 6-iodoacetamidofluorescein, (b) showed a proteolytic susceptibility that was intermediate between native Cpn60 and Cpn60 in 2.5 M urea, and (c) were not able to capture a folding intermediate of the enzyme rhodanese. After incubation at atmospheric pressure, monomeric Cpn60 regained the ability to interact with rhodanese intermediates, and the sulfhydryl reactivity fell before significantly reassociating to 14-mers. The different rates of recovery of the native properties indicate that a complex series of conformational events occur following depressurization. Finally, the monomers resulting from pressure were different from those produced from Cpn60 by the action of 2.5 M urea. These results demonstrate that there is a fast, pressure-induced dissociation of the Cpn60 14-mer followed by a conformational drift of the dissociated monomers that can be influenced by the presence of MgATP.

Adenosine Triphosphate↗
PubMed1995

Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer.

The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and competent to participate in protein folding are formed by low concentrations of urea (< 2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecules of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. These monomers display limited proteolysis by chymotrypsin at several points within a hydrophobic sequence centered around residue 250 to produce a relatively stable N-terminal fragment (approximately = to 26 kDa) and a partially overlapping C-terminal fragment (approximately = to 44 kDa). The exposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn60. For example, subdenaturing concentrations of guanidinium chloride (< or = 0.75 M) or the stabilizing salt, guanidinium sulfate, as well as NaCl or KCl are effective. The trivalent cation, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfaces within a charged context, would be particularly effective in binding to Cpn60.

Amino Acid Sequence↗
PubMed1995

Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains.

NADPH-cytochrome P450 reductase (reductase) contains FMN and FAD in 1:1 stoichiometry as tightly bound cofactors. Electrons from NADPH are transferred to cytochrome P450 through the intermediacy of reductase. A knowledge of the interactions which must occur to allow the intermolecular and intramolecular transfer of electrons is not only of intrinsic interest but is necessary to understand the regulation of the overall oxidation-reduction processes in which cytochromes P450 participate in the endoplasmic reticulum of many organs. In the present study, urea has been employed as a chaotropic agent to study the dissociation of flavins from NADPH-cytochrome P450 reductase. The results show that dissociation of FMN occurs at concentrations of urea between 0 and 1 M and that, as the concentrations of urea approach 1 M, the intrinsic protein fluorescence increases, indicating a change in protein conformation. Above 2 M urea protein fluorescence increases, reaching a plateau at 3 M urea, and FAD begins to dissociate from the enzyme. In the range of 0-1 M urea, a completely reversible dissociation of FMN occurs and, at 3 M urea, the fluorescence values representing flavin dissociation and protein conformation changes have reached a maximum. Thus, the definition of various states of the flavoprotein with both, one, or no flavins bound and the ability to remove the flavins reversibly under specific conditions have permitted the construction of a simple model to explain the various unfolding intermediates of this enzyme. Our experiments suggest that reductase is composed of distinct domains which can be examined independently by the application of chaotropic agents.

Circular Dichroism↗
PubMed1995

Chinese hamster rhodanese cDNA: activity of the expressed protein is not blocked by a C-terminal extension.

We describe a cDNA from Chinese hamster ovary cells which encodes a protein 91 and 96% identical to bovine and rat mitochondrial rhodaneses, respectively. Recombinant protein was expressed from the cDNA in Escherichia coli, purified to homogeneity, and found to have kinetic properties nearly indistinguishable from those of the bovine enzyme, the only cloned rhodanese previously verified by characterization of the recombinant protein. The carboxyl-terminus of the enzyme is characterized by a duplicated tripeptide, which can be proteolytically processed in vivo. We constructed a mutant in which the last 5 amino acids were replaced by 28 residues of unrelated sequence. This protein was expressed, purified, and found to have kinetic constants similar to those of the wild-type enzyme. The functionally verified Chinese hamster rhodanese cDNA encodes a protein of 297 residues and differs from the rat enzyme at 13 positions. None of these substitutions occurs at residues suggested to play essential roles in catalysis or structural stabilization.

Animals↗
PubMed1995

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Animals↗
PubMed1994

The stability of the molecular chaperonin cpn60 is affected by site-directed replacement of cysteine 518.

Cysteine 518 of the molecular chaperonin cpn60 (groEL) has been replaced with serine (C518S) by site-directed mutagenesis. The resulting mutant chaperonin protein is still functional and it can: (a) arrest the spontaneous folding of rhodanese in the absence of GroES and ATP, (b) assist refolding of the enzyme rhodanese in the presence of GroES and ATP/Mg2+, and (c) permit the urea-induced release and refolding of rhodanese from its complex with cpn60. ATP/Mg2+, alone, could discharge active rhodanese from cpn60 complexes formed with either wild type or C518S. In contrast with wild type cpn60, C518S has: (a) reduced stability of its quaternary structure, (b) reduced ability to reassemble tetradecamers after dissociation by urea; (c) reduced ATPase activity; and (d) more easily exposed hydrophobic surfaces. The data suggest that replacement of Cys-518 with Ser in cpn60 destabilizes its oligomeric structure, but there is no significant effect on cpn60 function or the stability of the monomers formed in urea.

Adenosine Triphosphatases↗
PubMed1994

Rapid secretion by a nonclassical pathway of overexpressed mammalian mitochondrial rhodanese.

Rhodanese is a small (33 kDa) monomeric sulfurtransferase which is synthesized on cytoplasmic ribosomes and imported into the mitochondrial matrix without cleavage of its amino terminus. When we transfected mammalian cell lines with rhodanese cDNA under the control of an efficient viral promoter, up to 40% of the overexpressed protein was secreted into the medium. This secretion was not the result of cell lysis, did not occur via the endoplasmic reticulum, and did not require the amino-terminal mitochondrial import signal. Addition of a carboxyl-terminal peptide extension did not block secretion, nor did a number of inhibitors of cellular sorting processes. Rhodanese polypeptide is known to associate with chaperonin proteins. In the absence of available mitochondrial import sites, such a complex in the cytoplasm of transfected cells could deliver unfolded rhodanese to export pores on the inner surface of the plasma membrane. This mechanism could contribute to the nonclassical secretion of cytoplasmically synthesized interleukins, growth factors, and lectins.

Amino Acid Sequence↗
PubMed1994

Bound substrate polypeptides can generally stabilize the tetradecameric structure of Cpn60 and induce its reassembly from monomers.

We demonstrate that the previously observed stabilizing effect by the enzyme rhodanese of the oligomeric structure of Cpn60 is general and can be provided by six other proteins that can interact with Cpn60. All these substrate proteins, which include examples that are monomeric, as well as oligomeric polypeptides in their native states, were shown previously to be assisted in their refolding by the chaperonin. Strikingly, during the disassembly of Cpn60 in the presence of any of the substrate proteins, significant amounts of intermediates were detected. Furthermore, unfolded substrate proteins induce the reassembly of tetradecameric Cpn60 from monomers, and binding of each substrate protein stabilizes Cpn60 quaternary structure.

Chaperonin 60↗
PubMed1994

Protein refolding at high concentrations using detergent/phospholipid mixtures.

Refolding of the sulfurtransferase enzyme rhodanese (EC 2.8.1.1) at high concentrations (0.2 mg/ml) was obtained at significant yields (> or = 45%) by using mixtures composed of detergents (either Triton X-100 or lauryl maltoside) and phospholipids. It is presumed that the ratio of detergent to phospholipid used in these mixtures results in the formation of very large micellar structures. Protein folding at lower concentrations (0.02 mg/ml) gave high yields (94%) when using the Triton X-100/phosphatidylglycerol micelle and these yields were higher than those obtained with either detergents or chaperonins. Differences between mixed micelle and liposome-mediated protein folding were clarified. We postulate that the method described here is successful at preventing misfolding and/or aggregation, and promoting correct folding, because the mixed micelle has both polar and nonpolar moieties which can bind to various exposed interactive sites in either unfolded proteins or protein folding intermediates. In this report, we detail the factors that allow the recovery of large amounts of active protein. This methodology should facilitate the large-scale production of biologically active, medically important proteins.

Animals↗
PubMed1994

Cysteine 254 can cooperate with active site cysteine 247 in reactivation of 5,5'-dithiobis(2-nitrobenzoic acid)-inactivated rhodanese as determined by site-directed mutagenesis.

Sulfhydryl substitution mutants of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) were used to determine whether the 4 cysteine residues in the native structure could cooperate in reactions. The sulfhydryl reactivity of persulfide-containing (ES) rhodanese was not significantly changed when cysteine residues at positions 63, 254, and 263 were replaced by serine, either individually or in combination. However, the sulfhydryl reactivity of persulfide-free (E) rhodanese was enhanced when Cys-254 was mutated. One sulfhydryl group, presumably the active site Cys-247, reacted rapidly in the E forms of these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4'-dipyridyl disulfide (4-PDS). After reaction with DTNB or 4-PDS, proteins with Cys-254 retained > 95% of their original activities as compared with Ser-254-containing proteins, which retained < 6% of their activities. Cyanide treatment could release thionitrobenzoate from rhodanese-thionitrobenzoate complexes with an approximate 1:1 stoichiometry. After this treatment, only the wild-type and C263S enzymes were fully active. Cyanide-treated rhodanese-thionitrobenzoate complexes of the C254S and C254S/C263S mutants could be fully reactivated using an exogenously added thiol, beta-mercaptoethanol. These results are consistent with the formation of a Cys-247-thiocyano derivative that is inactive but capable of being reactivated by intramolecular transfer of cyanide to Cys-254. In the absence of Cys-254, beta-mercaptoethanol can serve as the transferring sulfhydryl group.

Animals↗
PubMed1994

Mutation of cysteine 254 facilitates the conformational changes accompanying the interconversion of persulfide-substituted and persulfide-free rhodanese.

Mutants of rhodanese (EC 2.8.1.1) that substitute Ser for Cys-254 and/or Cys-263, i.e. C254S, C263S, and the double mutant C254/263S, were compared with the wild-type enzyme to test the hypothesis that the persulfide formation to give sulfur-substituted rhodanese (ES) during catalysis can exert a stabilizing influence on the global structure of the protein. All the ES forms were quite refractory toward limited tryptic digestion. Extensive digestion was observed only in the persulfide-free forms (E) of mutants having substitutions of Ser for Cys-254 (C254/263S and C254S). Limited proteolysis of C254/263S produced a species on SDS gels at approximately 21 kDa that was proteolyzed further to give a slightly smaller product. Amino acid sequencing, together with mass spectrometry, indicated that these two components had the same N-terminal sequence as wild-type rhodanese and that they resulted from cleavage in the C-terminal domain, first at residues Arg-186 or Arg-182 and subsequently at Arg-175 or Lys-174. In addition to increased proteolytic susceptibility, the conversion from ES to E of C254/263S was accompanied by increased hydrophobic exposure and increased quenching of the intrinsic protein fluorescence by acrylamide. The substitution of Ser for Cys-254, within the helix suggested to be involved in stabilizing the persulfide bond in ES, seems to promote the formation of flexible conformations of sulfur-free rhodanese. In keeping with this model, C254/263S shows a marked increase in the hysteresis that is observed in rhodanese catalysis at pH 7 and high ionic strength. These results can be understood in terms of a model, suggesting that there is mutual stabilization of the protein structure and the persulfide bond in the ES form of rhodanese.

Amino Acid Sequence↗