Interaction of trigger factor with the ribosome

The trigger factor of Escherichia coli is a prolyl isomerase and a chaperone. It interacts with the ribosome and affects the folding of newly formed protein chains. Therefore, the dynamics of the interactions of trigger factor with the ribosome and with unfolded protein chains should be tailored for this function. Previously, we had found that binding of unfolded proteins to trigger factor is fast and that the lifetime of the complex between these two components is only about 100 ms. Here, we have labeled the trigger factor in its amino-terminal, ribosome-binding domain with a fluorescent dye and investigated how it interacts with the ribosome. We found that this association, as well as the dissociation of the complex, are rather slow processes. The average lifetime of the complex is about 30 seconds (at 20 degrees C). The strong differences in the dynamics of the interactions of trigger factor with the ribosome and with protein substrates might ensure that, on the one hand, trigger factor remains bound to the ribosome while a protein chain is being synthesized, and, on the other hand, allows it to scan the newly formed protein for prolyl bonds that need catalysis of isomerization.     

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator

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Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.     

Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

GroEL walks the fine line: the subtle balance of substrate and co-chaperonin binding by GroEL. A combinatorial investigation by design, selection and screening

While support in protein folding by molecular chaperones is extremely efficient for endogenous polypeptides, it often fails for recombinant proteins in a bacterial host, thus constituting a major hurdle for protein research and biotechnology. To understand the reasons for this difference and to answer the question of whether it is feasible to design tailor-made chaperones, we investigated one of the most prominent bacterial chaperones, the GroEL/ES ring complex. On the basis of structural data, we designed and constructed a combinatorial GroEL library, where the substrate-binding site was randomized. Screening and selection experiments with this library demonstrated that substrate binding and release is supported by many variants, but the majority of the library members failed to assist in chaperonin-mediated protein folding under conditions where spontaneous folding is suppressed. These findings revealed a conflict between binding of substrate and binding of the co-chaperonin GroES. As a consequence, the window of mutational freedom in that region of GroEL is very small. In screening experiments, we could identify GroEL variants slightly improved for a given substrate, which were still promiscuous. As the substrate-binding site of the GroEL molecule overlaps strongly with the site of cofactor binding, the outcome of our experiments suggests that maintenance of cofactor binding affinity is more critical for chaperonin-mediated protein folding than energetically optimized substrate recognition.     

Cloning of cDNAs for Cecropins A and B,and Expression of the Genes in the Silkworm,Bombyx mori

cDNA clones coding cecropins A and B were isolated from a cDNA library constructed from the fat body of immunized Bombyx mori larvae. The cloned cDNAs had an open reading frame of 63 amino acids, indicating the primary translated peptides were processed to form mature cecropins of 35 amino acid residues. The homology in the coding regions of cecropins A and B was 73%.

In immunized fat body, the expression of both cecropin A and B genes reached the maximal level 5 h after the injection of soluble peptidoglycan, and the high level was maintained until 9 h after immunization. The cecropin A and B genes were expressed at high levels in fat body and hemocytes, at lower but significant levels in malpighian tube, slightly in midgut, and none in silk gland.     

Chimeric Antimicrobial Peptides Exhibit Multiple Modes of Action

Pyrrhocoricin and drosocin, representatives of the short, proline-rich antimicrobial peptide family kill bacteria by inactivating the bacterial heat shock protein DnaK and inhibiting chaperone-assisted protein folding. The molecular architecture of these peptides features an N-terminal DnaK-binding half and a C-terminal delivery unit, capable of crossing bacterial membranes. Cell penetration is enhanced if multiple copies of pyrrhocoricin are conjugated. To obtain drug leads with improved antimicrobial properties, and possible utility as therapeutic agents, we synthesized chimeric dimers, in which pyrrhocoricins potent DnaK-binding domain was connected to drosocins superior cell penetrating module. Indeed, the new constructs not only exhibited enhanced in vitro antibacterial properties against the originally sensitive strains Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, but also showed activity against Staphylococcus aureus, a bacterial strain resistant to native pyrrhocoricin and drosocin. The improved antimicrobial profile could be demonstrated with assays designed to distinguish intracellular or membrane activities. While a novel mixed pyrrhocoricin–drosocin dimer and the purely pyrrhocoricin-based old dimer bound E. coli DnaK with an identical 4 M K_d, the mixed dimers penetrated a significantly larger number of E. coli and S. aureus cells than the previous analogs and destroyed a larger percentage of bacterial membrane structures. Toxicity to human red blood cells could not be observed up to the highest peptide concentration tested, 640 M. In addition, repetitive reculturing of E. coli or S. aureus cells with sublethal concentrations of the mixed dimer did not result in resistance induction to the novel peptide antibiotic. The new concept of pyrrhocoricin–drosocin mixed dimers yields antibacterial peptide derivatives acting with a multiple mode of action, and can serve as a useful addition to the current antimicrobial therapy repertoire.     

Protease pro region required for folding is a potent inhibitor of the mature enzyme.

alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Elevated oxalate oxidase activity is correlated with Al-induced plasma membrane injury and root growth inhibition in young barley roots

In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several (3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting its role in oxidative stress and subsequent cell death caused by Al toxicity in plants.     

Predicting the structures of 18 peptides using Geocore

We describe an extensive test of Geocore, an ab initio peptide folding algorithm. We studied 18 short molecules for which there are structures in the Protein Data Bank; chains are up to 31 monomers long. Except for the very shortest peptides, an extremely simple energy function is sufficient to discriminate the true native state from more than 10(8) lowest energy conformations that are searched explicitly for each peptide. A high incidence of native-like structures is found within the best few hundred conformations generated by Geocore for each amino acid sequence. Predictions improve when the number of discrete phi/psi choices is increased.     

Identification and characterization of folding inhibitors of hen egg lysozyme: an example of a new paradigm of drug design

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme.     

Translocons, thermodynamics, and the folding of membrane proteins

Recent three-dimensional structures of helical membrane proteins present new challenges for the prediction of structure from amino acid sequence. Membrane proteins reside stably in a thermodynamic free energy minimum after release into the membrane's bilayer fabric from the translocon complex. This means that structure prediction is primarily a problem of physical chemistry. But the folding processes within the translocon must also be considered. A distilled overview of the physical principles of membrane protein stability is presented, and extended to encompass translocon-assisted folding.     

The beta-amyloid peptide of Alzheimer's disease decreases adhesion of vascular smooth muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Abeta) builds up. In this study, the possibility that Abeta disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Abeta in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Abeta was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Abeta that was fluorescently labelled at the N-terminus (fluo-Abeta) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Abeta. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Abeta1-40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Abeta treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Abeta on cell adhesion. The studies suggest that Abeta can decrease VSMC viability by disrupting VSMC-extracellular matrix (ECM) adhesion.     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.