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R Seckler

Publications and source records attributed to R Seckler.

At least 37 records · Page 2Linked to original sources
PubMed1993

Bacterial protein disulfide isomerase: efficient catalysis of oxidative protein folding at acidic pH.

Periplasmic protein disulfide isomerase (DsbA) is essential for disulfide formation in Escherichia coli. The tryptophan fluorescence of DsbA measures the redox state of the enzyme during catalysis of the oxidative folding of hirudin, a thrombin inhibitor containing three disulfide bonds and lacking tryptophan. With stoichiometric amounts of DsbA, reduced hirudin is rapidly oxidized in a process initially leading to random disulfides. Disulfide reshuffling involving reduced DsbA yields completely native inhibitor within 1 h, even at pH 4. Catalytic amounts of DsbA become essential for hirudin folding in the presence of redox buffers at acidic pH. The second-order rate constants of disulfide exchange reactions involving DsbA are several orders of magnitude above the known values for alkyl dithiols and disulfide oxidoreductases. DsbA preferably reacts with reduced, unfolded polypeptides. The reduction of DsbA by hirudin is faster by 1 order of magnitude than its reduction by the strong reductant dithiothreitol. Together, unusually fast disulfide interchange reactions and a preference for folding polypeptides appear to be responsible for the catalytic efficiency of DsbA and for disulfide formation in vivo at acidic pH.

Amino Acid Sequence↗
PubMed1993

Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro.

One of the central problems in protein folding is how amino acid sequences within polypeptide chains direct polypeptide chain folding and avoid off-pathway aggregation both in intracellular environments and in the test tube. The tailspike protein of phage P22 is a model system for which genetic analysis has permitted mutational dissection of the role of amino acid positions in the polypeptide chain in directing its in vivo folding. Two classes of mutations that affect intracellular folding and aggregation have been characterized; temperature-sensitive folding (tsf) mutants and second-site suppressors of tsf mutants. Here we report the effects of these mutations on the in vitro refolding and aggregation pathway of the purified proteins. The tsf mutations reduced refolding yields at high temperature and increased aggregation, while second-site suppressors enhanced refolding and inhibited aggregation in the test tube. For both types of mutations, the strength of the effects observed in vitro correlated with their in vivo phenotypes. The results confirm that the mutations act intrinsically on the folding pathway of the tailspike polypeptide and not through accessory proteins.

Amino Acid Sequence↗
PubMed1993

Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain.

Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N-terminally shortened protein fragment comprising amino-acid residues 108-666 [Chen, B.-L. & King, J. (1991) Biochemistry 30, 6260-6269]. In the present work, the 60-kDa C-terminal fragment was purified to homogeneity from the tryptic digest by gel-fitration chromatography. As in the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS-resistant, enzymatically active trimer. However, its unfolding in the presence of guanidinium chloride was accelerated at least 10-fold compared to the complete, native tailspike protein. Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid-urea-unfolded fragment polypeptides with neutral buffer. Upon recombinant expression of the 60-kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS-resistant trimers. The refolding and assembly pathway of the N-terminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels. As found for the complete tailspike protein, yields of refolding and assembly of the 60-kDa fragments into SDS-resistant trimers decreased with increasing temperature. The refolding yield of fragments derived from a temperature-sensitive mutant (Gly244-->Arg) tailspike protein was affected in similar fashion as shown for the whole protein. We conclude that the N-terminal domain (residues 1-107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1993

Interactions of phage P22 tailspike protein with GroE molecular chaperones during refolding in vitro.

Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated. At elevated temperature (> 30 degrees C), in the absence of ATP, GroEL effectively trapped refolding tailspike protein and prevented reconstitution. Tailspike protein was released from GroEL by addition of ATP around 35 degrees C or without added ATP upon cooling to 25 degrees C, and native tailspike trimers were formed. In accordance with the cold release, tailspike reconstitution at < or = 25 degrees C was unaffected by GroE. No formation of native tailspike trimers was observed, when refolding was initiated at 42 degrees C in the presence of the GroE proteins and ATP or when tailspike protein was dissociated from a preformed complex with the chaperone by addition of ATP at 42 degrees C. In contrast to other GroE ligands, the tailspike polypeptide was bound by and released from GroE in similar states of folding, and the presence of GroES in addition to GroEL had no effect on reconstitution yields at any temperature. Thus, the GroE proteins may exhibit widely differing interactions even with proteins showing similarly temperature-sensitive yields of folding.

Bacterial Proteins↗
PubMed1993

Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Reactivators↗
PubMed1993

Mechanism of phage P22 tailspike protein folding mutations.

Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.

Bacteriophage P22↗
PubMed1992

Protein folding and protein refolding.

The functional three-dimensional structure of proteins is determined solely by their amino acid sequences. Protein folding occurs spontaneously beginning with the formation of local secondary structure concomitant with a compaction of the molecule. Secondary structure elements subsequently interact to form subdomains and domains stabilized by tertiary interactions. Disulfide bond formation, and cis-trans isomerization of X-Pro peptide bonds, as the rate-limiting folding reactions, are enzymatically catalyzed during protein folding in the cell. Although folding of domains is fast enough to occur cotranslationally in vivo, such vectorial folding on the ribosome is not essential for attainment of the native structure of a protein. Slow steps on the pathway to the functional protein structure are docking reactions of domains, association of subunits, or reshuffling reactions at the oligomer level. Aggregation as a competing side reaction is prevented, and the kinetic partition between competing polypeptide folding and translocation reactions is regulated by chaperone proteins binding to incompletely folded polypeptides.

Animals↗
PubMed1991

Characterization, stability and refolding of recombinant hirudin.

A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.

Chromatography, High Pressure Liquid↗
PubMed1991

In vitro folding pathway of phage P22 tailspike protein.

The intracellular chain folding and association pathway of the thermostable, trimeric phage P22 tailspike endorhamnosidase has been the subject of a previous detailed study employing temperature-sensitive folding mutants. Recently, reconstitution of native tailspikes from completely unfolded polypeptides has been accomplished, providing a model system to compare protein folding pathways in vivo and in vitro. The in vitro reconstitution pathway of the protein after dilution from guanidine hydrochloride or acid-urea solutions at 10 degrees C was characterized by spectroscopic and hydrodynamic techniques, and may be summarized as an ordered sequence of folding, association, and folding reactions. Multiphasic folding of monomers was indicated by changes in circular dichroism and fluorescence, with a rate constant of k = 1.6 X 10(-3) s-1 for the slowest phase observed spectroscopically. Trimerization of structured monomers was followed by size-exclusion HPLC and was completed within 1.5 h at a protein concentration of 20 micrograms/mL. Although at this time trimers did not exchange subunits, they were readily dissociable by dodecyl sulfate in the cold. Formation of native, detergent-resistant trimers was only completed after 3 days of reconstitution at 10 degrees C. The reconstitution pathway of the tailspike protein closely resembles its intracellular maturation path. Thus, the in vitro reconstitution system, as a valid model of chain folding and association in vivo, should provide the tools to localize the steps or intermediates on the pathway that are the targets of temperature-sensitive folding mutations.

Chromatography, High Pressure Liquid↗
PubMed1991

Intermolecular interactions involved in the association of the variant surface glycoprotein of Trypanosoma brucei.

Trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (VSG). Depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. VSG is accessible as soluble globular protein (sVSG), or as insoluble membrane form (mfVSG). In order to get insight into the two-dimensional association of VSG within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. No self-assembly of sVSG could be detected even at protein concentrations close to the local packing in the surface layer. The absence of preferential interactions with soybean phospholipid or lysolecithin monolayers (spread on a Langmuir trough) suggests that the soluble form of the protein is not integrated into a model lipid-water interface. Thus, the two-dimensional arrangement of the protein in situ seems to be determined by hydrophobic interactions of the lipid components rather than protein-lipid interactions. In contrast to sVSG, the membrane form (mfVSG) undergoes aggregation and shows a strong tendency to absorb to surfaces and chromatographic matrices, thus interfering with standard techniques of protein purification.

Animals↗
PubMed1990

Stability and reconstitution of the soluble variant surface glycoprotein (sVSG) from Trypanosoma brucei.

Soluble variant surface glycoprotein (sVSG) is the form of the coat protein of Trypanosoma brucei released by cleavage of its lipid anchor. As shown by ultracentrifugal analysis, the protein of the variant clone MITat 1.2 is a stable dimer of (117 +/- 6)-kDa molecular mass. Its quaternary structure remains unaltered in the concentration range from 0.01 to approximately 50 mg/mL. Further extrapolation to the in situ concentration on the cell surface points to no significant protein association beyond the dimer, because after correction for solution viscosity sedimentation velocity is independent of the protein concentration. The sedimentation constant, s20,w = 5.1 X 10(-13) s, together with the particle weight confirms the high anisotropy of the dimer. Circular dichroism and fluorescence spectra show the typical properties of an alpha-helical protein (51% alpha-helix) with fluorophores buried in the hydrophobic interior of the protein. Denaturation at extremes of pH leads to the monomer still maintaining a relatively compact structure. Increased concentrations of urea and guanidine hydrochloride cause randomization with cooperative transitions at 1.7 and 0.7 M, respectively. The yield of reconstitution of the denatured protein reaches 87% under optimum conditions. The final product is indistinguishable from the native protein in its spectral, hydrodynamic, and immunochemical properties. Immunological analysis included polyclonal antibodies as well as monoclonal antibodies raised against epitopes in the surface of the complete trypanosome, as well as cryptic epitopes exposed only on sVSG in solution. The kinetics of reconstitution involve sequential uni-bimolecular processes, corresponding to consecutive folding and subunit association. About 38% of the fluorescence of the native protein is recovered within the mixing time (secondary structure formation).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex.

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.

Animals↗
PubMed1989

Reconstitution of the thermostable trimeric phage P22 tailspike protein from denatured chains in vitro.

Intermediates in the intracellular chain folding and association pathway of the P22 tailspike endorhamnosidase have been identified previously by physiological and genetic methods. Conditions have now been found for the in vitro refolding of this large (Mr = 215,000) oligomeric protein. Purified Salmonella phage P22 tailspikes, while very stable to urea in neutral solution, were dissociated by moderate concentrations of urea at acidic pH. The tailspike protein was denatured to unfolded polypeptide chains in 6 M urea, pH 3, as disclosed by analytical ultracentrifugation, fluorescence, and circular dichroism. Upon dilution into neutral buffer at 10 degrees C, the polypeptides fold spontaneously and associate to form trimeric tailspikes with high yield. Like native phage P22 tailspikes, the reconstitution product is resistant to denaturation by dodecyl sulfate in the cold and displays endorhamnosidase activity. Sedimentation coefficients, electrophoretic mobility, and fluorescence emission maxima of native and reconstituted tailspikes are identical within experimental error. By characterization of intermediates, localization of temperature-sensitive steps, and analysis of the effect of previously identified folding mutations, the reconstitution system described should allow comparison of in vivo and in vitro folding pathways of this large protein oligomer.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

Amino Acid Sequence↗
PubMed1986

Two adjacent epitopes on a synthetic dodecapeptide induce lactate dehydrogenase B-specific helper and suppressor T cells.

The outcome of an immune response to the enzyme lactate dehydrogenase B (LDH-B) is determined by the interplay between two types of regulatory T lymphocytes, T helper (Th) and T suppressor (Ts) cells. Most mouse strains are capable of generating Th but not Ts cells, and are therefore high responders to LDH-B in terms of both antibody production and antigen-specific T-cell proliferation. However, in strains expressing the b or k allele at the E beta locus of the major histocompatibility complex (Mhc), Ts cells are induced that partly or totally abrogate the proliferative response of Th cells to LDH-B. As a result, these strains are phenotypically medium (E beta b expressors) or low (E beta k expressors) responders. Because the suppression in the LDH-B system is antigen-specific (i.e. it only affects LDH-B-specific Th cells), it is conceivable that the Th and Ts cells use the antigen itself to communicate with each other. To investigate this possibility, we set out to determine which epitopes of the LDH-B molecule are recognized by Th and Ts cells. On the basis of previous studies, a loop structure extending from residue 211 to residue 224 of pig LDH-B appeared to be preferentially recognized by most Th-type (class II Mhc-restricted, proliferating) clones. By using a synthetic peptide, we demonstrate here that both Th and Ts cells are induced by the 211-222 stretch of LDH-B sequence. The use of two further dodecapeptides, each with a single amino-acid substitution in comparison with the pig 211-222 sequence, has revealed that Th and Ts cells have different fine specificities. Thus the loop appears to have two closely linked, if not overlapping, epitopes, one recognized by Th and the other by Ts cells. This finding is consistent with two possible mechanisms of suppression, namely bridging of Th and Ts cells by antigen and subsequent transmission of a suppressive signal, and competition for antigen between Th and Ts cells.

Animals↗
PubMed1986

Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane.

From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits. Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein. Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies. Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane. Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.

Amino Acid Sequence↗
PubMed1986

Quantitative immunoprecipitation of the lactose transporter from Escherichia coli confirms stoichiometry of substrate binding.

A procedure is presented for the immunoprecipitation of the E. coli lactose transporter which may be applicable to other membrane protein antigens. Antibodies to its chemically synthesized C-terminal decapeptide specifically precipitate 6.0 +/- 0.2% of the radioactivity from solubilized [14C]amino-acid-labeled total cell envelopes of the transporter-overproducing strain T206 corresponding to 1.0 +/- 0.1 nmol transporter/mg total membrane protein. Comparison with galactoside binding yields a stoichiometry of 1.1 +/- 0.2 mol galactoside bound/mol transporter.

Chemical Precipitation↗