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1.
Abstract In the presence of phenethylalcohol, plasmidencoded levansucrase (2,6β- d -fructan: 6,β- d -fructosyltransferase, EC 2.4.1.10) was accumulated as a precursor form in the membrane fraction of Escherichia coli minicells. Immunoblotting experiments were used to evaluate the amount of the newly synthesised precursor form relative to the immunoreactivity of the purified mature form of levansucrase. Solubilisation of both forms with Triton X100 and Triton X114 was compared. Triton X114 allowed separation of the precursor form from the mature form, which were recovered in the detergent and the aqueous phases, respectively, after separation of the phases at 30°C. 相似文献
2.
The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion 总被引:1,自引:0,他引:1
Marie-Françoise Petit-Glatron Lydie Grajcar Anette Munz Régis Chambert 《Molecular microbiology》1993,9(5):1097-1106
A weak Ca2+-binding site (Ka= 0.8× 103 M?1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca2+ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process. 相似文献
3.
Secretion of Bacillus subtilis levansucrase. Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes. 总被引:1,自引:0,他引:1
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The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events. 相似文献
4.
The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly
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Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria. 相似文献
5.
The precursor of beta-lactamase: purification, properties and folding kinetics. 总被引:12,自引:5,他引:7
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The precursor of Escherichia coli RTEM beta-lactamase was purified to homogeneity on a milligram scale by a procedure independent of the binding properties of the protein and refolded to an active, reduced form. For comparing the folding kinetics, the wild-type enzyme was reduced and a mutant was constructed, in which the two cysteines that form a very stable disulfide bond in the RTEM enzyme were both changed into alanines. The rate of folding was determined by directly measuring the increase in enzymatic activity. The reduced precursor folds at least 15 times more slowly than either the reduced mature enzyme or the mature Cys----Ala double mutant under identical conditions. The wild-type enzyme, the Cys----Ala double mutant and the precursor protein all had similar KM values, demonstrating a very similar native state. The slow folding of the precursor compared with the mature form may be an essential and general feature to secure a transport competent conformation necessary for the translocation through a membrane in protein export. This folding assay of a precursor by directly following its enzymatic activity may facilitate the characterization of putative folding modulators in bacterial membrane transport. 相似文献
6.
Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein. 相似文献
7.
Sílvia Bronsoms Josep Villanueva Francesc Canals Enrique Querol Francesc X Aviles 《European journal of biochemistry》2003,270(17):3641-3650
Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form. 相似文献
8.
Yukihiro Yabuta Ezhilkani Subbian Hiroshi Takagi Ujwal Shinde Masayori Inouye 《Journal of biochemistry》2002,131(1):31-37
Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca(2+) binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca(2+) ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca(2+) ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links alpha-helix-2 and beta-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca(2+) binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency. 相似文献
9.
Lázaro Hernández Juan Arrieta Lázaro Betancourt Viviana Falcón Joel Madrazo Alberto Coego Carmen Menéndez 《Current microbiology》1999,39(3):146-152
Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron
microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular
forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics
indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg
as determined by PSD-MALDI-TOFMS (post-source decay—matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry).
Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide
followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.
Received: 24 March 1999 / Accepted: 30 April 1999 相似文献
10.
Prosequence-mediated disulfide coupled folding of the peptide hormones guanylin and uroguanylin 总被引:1,自引:0,他引:1
In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein. 相似文献
11.
Conotoxins comprise a large and diverse group of peptide neurotoxins derived from Conus snail venoms; most contain multiple disulfide bonds. The conotoxin precursors consist of three distinct domains: the N-terminal signal sequence, an intervening propeptide region, and the C-terminal mature conotoxin. Formation of the native disulfide bonds during the oxidative folding of conotoxins is a prerequisite for their proper biological function, but in numerous in vitro folding experiments with mature conotoxins, a lack of specificity in formation of the native Cys-Cys connectivities is observed. The mechanisms that ensure that the native disulfide bonds are formed in venom ducts during biosynthesis remain unknown. To evaluate whether the propeptide could potentially function as an intramolecular chaperone, we studied the oxidative folding of a conotoxin precursor, pro-GI, belonging to the alpha-conotoxin family. Our results indicate that the propeptide sequence did not directly contribute to folding kinetics and thermodynamics. However, we found that the propeptide region of pro-GI played an important role when oxidative folding was catalyzed by protein disulfide isomerase (PDI). The PDI-assisted reaction was more efficient during the early folding in the context of the propeptide sequence (pro-GI), as compared to that of the mature conotoxin (alpha-GI). Taken together, our results suggest for the first time that the propeptide region may play a role in the PDI-catalyzed oxidative folding of conotoxin precursors. 相似文献
12.
Engineering-enhanced protein secretory expression in yeast with application to insulin 总被引:2,自引:0,他引:2
Kjeldsen T Ludvigsen S Diers I Balschmidt P Sorensen AR Kaarsholm NC 《The Journal of biological chemistry》2002,277(21):18245-18248
Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp(B28)) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin. 相似文献
13.
Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli 总被引:7,自引:0,他引:7
High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro. 相似文献
14.
Levansucrase of Bacillus subtilis: effects on the secretion process of single amino acid substitutions in the mature part of the protein 总被引:1,自引:0,他引:1
Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of Bacillus subtilis levansucrase were obtained by mutagenesis. Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein. The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins. Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced. Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme. The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process. 相似文献
15.
Bacillus subtilis levansucrase: amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals 总被引:6,自引:1,他引:5
Studies of the equilibrium between native and denatured forms of wild-type levansucrase showed that the denatured form was predominant at 37 degrees C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal-dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly-366----Asp or Gly-366----Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly-366----Ser variant was secreted at near-normal levels and shared a part of the in vitro refolding properties of the wild-type protein. These differential properties might be related to the ability of the altered region to form a beta-turn structure. We discuss the possible role of metal ions in the coupling of protein folding and secretion. 相似文献
16.
Kang HJ Kim JH Chang WJ Kim ES Koo YM 《Journal of microbiology and biotechnology》2007,17(11):1751-1757
Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification. 相似文献
17.
Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. 总被引:19,自引:0,他引:19
The transport of precursor proteins into mitochondria requires an energized inner membrane. We report here that the import of various precursor proteins showed a differential sensitivity to treatment of the mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The differential inhibition by carbonyl cyanide m-chlorophenylhydrazone was not influenced by the length of the precursor, the presence of mature protein parts, or the folding state of the precursor but was specific for the presequence. Moreover, only the membrane potential delta psi and not the total proton motive force was required for the transport of precursors, indicating that protein translocation across the inner membrane is not driven by a movement of protons. We conclude that delta psi (negative inside) is needed for the translocation of the positively charged presequences, possibly via an electrophoretic effect. 相似文献
18.
Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. 相似文献
19.
A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3' terminal codon of sacB, the structural gene of levansucrase. This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase. The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50-55% of that found for the wild-type levansucrase (20 mg l-1). The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase. However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded. 相似文献
20.
Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention. 相似文献