Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV

Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.     

Release of hydrophobic anticancer drug from a newly designed self-assembling peptide

A newly designed self-assembling peptide, P4 (Ac-NH-LDLKLELKLDLKLELK-CONH(2)), capable of stabilizing hydrophobic compounds in aqueous solution has been discovered. The ionic self-complementary peptide P4 has 16 amino acids, ～5 nm in size, with an alternating polar and non-polar pattern. Circular dichroism analysis demonstrated that P4 forms stable β-sheet structures, and self-assembles into nanofibers, which was demonstrated by atomic force microscopy. These nanofibers can form a scaffold hydrogel consisting of >99% water. It showed that the P4 hydrogel had stable hydrogel rheological properties. The ability of the peptide to stabilize the hydrophobic anticancer agent ellipticine was tested in this research. The results showed that the state of ellipticine in the complexes was dependent on the concentration of the peptide, which also affected the size and morphology of the complex. The anticancer activity of the complexes was studied by testing the viability with a MTT assay and a LIVE/DEAD Viability/Cytotoxicity kit in two cancer cell lines including SMMC7721 and EC9706. The viability results showed that complexes of protonated ellipticine could significantly reduce the viability of the two cell lines. The results described herein provide further incentives for basic studies on self-assembling peptide-based delivery of hydrophobic anticancer drugs.     

Self-assembly of the ionic peptide EAK16: the effect of charge distributions on self-assembly

Amphiphilic peptides suspended in aqueous solution display a rich set of aggregation behavior. Molecular-level studies of relatively simple amphiphilic molecules under controlled conditions are an essential step toward a better understanding of self-assembly phenomena of naturally occurring peptides/proteins. Here, we study the influence of molecular architecture and interactions on the self-assembly of model peptides (EAK16s), using both experimental and theoretical approaches. Three different types of EAK16 were studied: EAK16-I, -II, and -IV, which have the same amino acid composition but different amino acid sequences. Atomic force microscopy confirms that EAK16-I and -II form fibrillar assemblies, whereas EAK16-IV forms globular structures. The Fourier transform infrared spectrum of EAK16-IV indicates the possible formation of a beta-turn structure, which is not found in EAK16-I and -II. Our theoretical and numerical studies suggest the underlying mechanism behind these observations. We show that the hairpin structure is energetically stable for EAK16-IV, whereas the chain entropy of EAK16-I and -II favors relatively stretched conformations. Our combined experimental and theoretical approaches provide a clear picture of the interplay between single-chain properties, as determined by peptide sequences (or charge distributions), and the emerging structure at the nano (or more coarse-grained) level.     

Pharmacokinetics of Peptide mediated delivery of anticancer drug ellipticine

The amino acid pairing peptide EAK16-II (EAK) has shown the ability to stabilize the hydrophobic anticancer agent ellipticine (EPT) in aqueous solution. In this study, we investigate pharmacokinetics of the formulation of EAK-EPT complexes in vivo. The developed formulation can achieve a sufficiently high drug concentration required in vivo animal models. The nanostructure and surface properties of EAK-EPT complexes or nanoparticle were characterized by transmission electron microscopy (TEM) and zeta potential measurements, respectively. 12 healthy male SD rats were divided into EPT group and EAK-EPT group randomly. Rats in EPT group were tail intravenously injected with the EPT (20 mg/kg); rats in EAK-EPT group were injected with EAK-EPT complexes (EPT's concentration is 20 mg/kg). EPT was extracted from rat plasma with dexamethasone sodium phosphate as internal standards (IS). The pharmacokinetic parameters were obtained using high pressure liquid chromatography (HPLC). Significant differences in main pharmacokinetic parameters between EPT and EAK-EPT complexes were observed, demonstrating that the complexation with EAK prolongs the residence time of the drug and enlarges the area under the concentration-time curve (AUC). This means that EAK can serve as a suitable carrier to increase the bioavailability of EPT.     

All-atom molecular dynamics study of EAK16 peptide: the effect of pH on single-chain conformation,dimerization and self-assembly behavior

Single-chain equilibrium conformation and dimerization of the three types of ionic EAK16 peptide are studied under three pH conditions using all-atom molecular dynamics simulations. It is found that both the single-chain conformation and the dimerization process of EAK16-IV are considerably different from those of the two other types, EAK16-I and EAK16-II. The value of pH is found to have a stronger effect on the single-chain conformation and dimerization of EAK16-IV. It is shown that in addition to the charge pattern on the peptide chains, the size of the side chains of the charged amino acids plays role in the conformation of the peptide chains and their dimerization. The results shed light on the pH-dependent self-assembly behavior of EAK16 peptide in the bulk solution, which has been reported in the literature.     

Modification of hydrophilic and hydrophobic surfaces using an ionic-complementary peptide

Ionic-complementary peptides are novel nano-biomaterials with a variety of biomedical applications including potential biosurface engineering. This study presents evidence that a model ionic-complementary peptide EAK16-II is capable of assembling/coating on hydrophilic mica as well as hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces with different nano-patterns. EAK16-II forms randomly oriented nanofibers or nanofiber networks on mica, while ordered nanofibers parallel or oriented 60 degrees or 120 degrees to each other on HOPG, reflecting the crystallographic symmetry of graphite (0001). The density of coated nanofibers on both surfaces can be controlled by adjusting the peptide concentration and the contact time of the peptide solution with the surface. The coated EAK16-II nanofibers alter the wettability of the two surfaces differently: the water contact angle of bare mica surface is measured to be <10 degrees , while it increases to 20.3+/-2.9 degrees upon 2 h modification of the surface using a 29 microM EAK16-II solution. In contrast, the water contact angle decreases significantly from 71.2+/-11.1 degrees to 39.4+/-4.3 degrees after the HOPG surface is coated with a 29 microM peptide solution for 2 h. The stability of the EAK16-II nanofibers on both surfaces is further evaluated by immersing the surface into acidic and basic solutions and analyzing the changes in the nanofiber surface coverage. The EAK16-II nanofibers on mica remain stable in acidic solution but not in alkaline solution, while they are stable on the HOPG surface regardless of the solution pH. This work demonstrates the possibility of using self-assembling peptides for surface modification applications.     

Mechanisms of the self-assembly of EAK16-family peptides into fibrillar and globular structures: molecular dynamics simulations from nano- to micro-seconds

The self-assembly of EAK16-family peptides in a bulk solution was studied using a combination of all-atom and coarse-grained molecular dynamics simulations. In addition, specified concentrations of EAK16 peptides were induced to form fibrillary or globular assemblies in vitro. The results show that the combination of all-atom molecular dynamics simulations on the single- and double-chain levels and coarse-grained simulations on the many-chain level predicts the experimental observations reasonably well. At neutral pH conditions, EAK16-I and EAK16-II assemble into fibrillary structures, whereas EAK16-IV aggregates into globular assemblies. Mechanisms of the formation of fibrillar and globular assemblies are described using the simulation results.     

Interaction of a self-assembling peptide with oligonucleotides: complexation and aggregation

Molecular interaction of a self-assembling peptide, EAK16-II, to single- and double-stranded oligodeoxynucleotides (ODNs) was investigated under various solution conditions. The molecular events leading to EAK-ODN complexation and further aggregation were elucidated using a series of spectroscopic and microscopic methods. Despite the ability to self-assemble, EAK molecules bind to ODN molecules first upon mixing, resulting in EAK-ODN complexes. The complexes further associate to form EAK-ODN aggregates. A method based on UV-Vis absorption and centrifugation was developed to quantify the fraction of ODNs in the aggregates. The results were used to construct binding isotherms via a binding density function analysis. To compare the effects of different pH values and nucleotide types, the modified noncooperative McGhee and von Hippel model was used to extract binding parameters from the binding isotherms. The binding constant of EAK to ODNs was higher at pH 4 than at pH 7, and no binding was observed at pH 11, indicating that the interaction involved is primarily electrostatic in nature. EAK bound more strongly to single-stranded ODNs. The EAK-ODN aggregates were further visualized using atomic force microscopy; their size distribution as a function of EAK concentration was monitored by dynamic light scattering. The timescale for the EAK-ODN aggregation was on the order of minutes by fluorescence anisotropy and steady-state light scattering experiments. Fluorescence quenching experiments demonstrated that the ODNs in the aggregates were less accessible to the solvent, demonstrating a potential of oligonucleotide encapsulation by the self-assembling peptide.     

Concentration effect on the aggregation of a self-assembling oligopeptide

Concentration is a key parameter in controlling the aggregation of self-assembling oligopeptides. By investigating the concentration effects, an aggregation mechanism of EAK16-II is proposed. Depending on the critical aggregation concentration (CAC) of EAK16-II, the oligopeptide aggregates into protofibrils through seeding and/or a nucleation process. Protofibrils then associate with each other to form fibrils. The CAC was found to be approximately 0.1 mg/ml by surface tension measurements. The nanostructures of aggregates were imaged and analyzed by atomic force microscopy. Globular and fibrillar aggregates were observed, and their dimensions were further quantified. To ensure that the aggregates were formed in bulk solution, light scattering (LS) measurements were conducted to monitor the fibril formation with time. The LS profile showed two different rates of aggregation depending on whether the peptide concentration was above or below the CAC. At high concentrations, the LS intensity increased strongly at early times. At low concentrations, the LS intensity increased only slightly. Our study provides information about the nature of the oligopeptide self-assembly, which is important to the understanding of the fibrillogenesis occurring in conformational diseases and to many biomedical engineering applications.     

Enrichment of fermentation media and optimization of expression conditions for the production of EAK(16) peptide as fusions with SUMO

EAK(16) (AEAEAKAKAEAKAEAK) belongs to a novel class of self-assembling peptides, which is being investigated in research and industry. SUMO belongs to the ubiquitin class of proteins and is a promising fusion partner currently in use. In this study, EAK(16) peptide fusions with hexa-histidine tagged SUMO have been constructed using Escherichia coli based pET expression vector. Intracellular expression of the SUMO-EAK(16) fusion using LB media has been optimized. Low-cost complex media (fungal autolysates, wheat and gluten hydrolysates) produced via a novel wheat-based biorefinery have been used as alternative fermentation media to LB. Shake flask cultures using either enriched LB or complex wheat-derived media containing 2 g/L of glucose resulted in intracellular SUMO-EAK(16) fusion protein production of approximately 250 mg/L fermentation volume which corresponded to 30-35% of the total bacterial protein expressed being the fusion protein. Fusion protein productivities up to five times higher were achieved when using a bioreactor.     

Morphology of artificial silica matrices formed via autosilification of a silaffin/protein polymer chimera

Protein polymers (long-chain proteins in which a specific amino acid sequence "monomer" is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK 1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK 1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.     

Effects of peptide secondary structure on the interaction with oppositely charged microgels

The importance of peptide secondary structure on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for EFKRIVQRIKDFLRNLV (EFK17). Through D-enantiomer (EFK17-d/a; E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) or tryptophan (EFK17-W/a; EWKRWVQRWKDFLRNLV) substitutions, both conformation-dependent and -independent amphiphilicity of this peptide could be precisely controlled. Peptide secondary structure was investigated by circular dichroism, whereas microgel deswelling and reswelling in response to peptide binding and release were studied by micromanipulator-assisted light and fluorescence microscopy, and peptide uptake in the microgels was determined from solution depletion measurements. Results show that peptide binding to the microgel is highly influenced by peptide secondary structure. EFK17-a, characterized by an idealized helix with all polar/charged amino acids located at one side of the helix, and all nonpolar/hydrophobic residues on the other, displays pronounced α-helix induction on peptide binding to the microgels. EFK17-d/a, on the other hand, displays no such amphiphilic helix induction. Mirroring this, EFK17-a displays substantially higher binding to the microgels than EFK17-d/a as well as much larger peptide-induced microgel deswelling. For EFK17-W/a, both conformation-dependent and -independent amphiphilicity effects were demonstrated. Overall, the results show that peptide conformational aspects need to be considered in peptide/microgel interactions, for example, in the design of microgel carrier systems for peptide drugs.     

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.     

Creating Prebiotic Sanctuary: Self-Assembling Supramolecular Peptide Structures Bind and Stabilize RNA

Any attempt to uncover the origins of life must tackle the known ‘blind watchmaker problem’. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.     

In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV

Protein translocation across the inner envelope of plastids is mediated by the TIC (translocon at the inner envelope membrane of chloroplasts) protein translocation machinery. Tic20 has been shown to function as a central component of TIC machinery. The Arabidopsis genome encodes four Tic20 homologous proteins, AtTic20-I, AtTic20-II, AtTIC20-IV and AtTic20-V, among which only AtTic20-I has been extensively characterized and demonstrated to be essential for protein import into chloroplasts. AtTic20-I is more closely related to AtTic20-IV than to AtTic20-II or AtTic20-V, whereas AtTic20-II and AtTic20-V show higher similarities to each other than to AtTic20-I or AtTic20-IV. Here, we show that AtTic20-IV is expressed mainly in roots whereas AtTic20-I is more abundant in shoots than in roots. Although AtTic20-IV is dispensable for viability in the wild-type background, interestingly, expression of AtTic20-IV is markedly elevated in both shoots and roots in the tic20-I knockout mutant that exhibits severe albino and seedling-lethal phenotypes. The albino tic20-I seedlings do not accumulate any of the photosynthetic proteins analyzed, but the plastids can still import non-photosynthetic housekeeping proteins. This residual import ability of the tic20-I mutant can be attributed to partial compensation by the elevated expression of AtTic20-IV, since a double knockout mutant of AtTic20-I and AtTic20-IV exhibits more severe embryonic lethality. Further overexpression of AtTic20-IV in the tic20-I mutant can only marginally rescue the accumulation of photosynthetic proteins in the albino seedlings. These data demonstrate an absolute requirement of at least one of the two closely related Tic20 proteins in protein translocation across the inner envelope of plastids and also suggest their distinct substrate preferences.     

Self-assembling peptide-polymer hydrogels designed from the coiled coil region of fibrin

Biomaterials constructed from self-assembling peptides, peptide derivatives, and peptide-polymer conjugates are receiving increasing attention as defined matrices for tissue engineering, controlled therapeutic release, and in vitro cell expansion, but many are constructed from peptide structures not typically found in the human extracellular matrix. Here we report a self-assembling biomaterial constructed from a designed peptide inspired by the coiled coil domain of human fibrin, the major protein constituent of blood clots and the provisional scaffold of wound healing. Targeted substitutions were made in the residues forming the interface between coiled coil strands for a 37-amino acid peptide from human fibrinogen to stabilize the coiled coil peptide bundle, while the solvent-exposed residues were left unchanged to provide a surface similar to that of the native protein. This peptide, which self-assembled into coiled coil dimers and tetramers, was then used to produce triblock peptide-PEG-peptide bioconjugates that self-assembled into viscoelastic hydrogel biomaterials.     

Folding and dimerization of the ionic peptide EAK 16-IV

Folding and dimerization of an ionic polyalanine-based peptide chain (EAK16-IV) are simulated with nonspecific interactions. It is found that there is a competition between two kinds of structural motifs under different strengths of electrostatic interactions. The dominance of hairpin-like structures would be realized with a strong electrostatic interaction both thermodynamically and kinetically, showing the importance of the electrostatic interaction on the formation of hairpin-like structures. Simulations on the dimerization with strong electrostatic interaction are also carried out. It is found that the concentration contributes essentially to the shape of the dimers. These studies demonstrate that the strong interactions and kinetic factors might be important for the ordered amyloid aggregates.     

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.     

Evidence that SecB enhances the activity of SecA

In Escherichia coli, SecA is a critical component of the protein transport machinery which powers the translocation process by hydrolyzing ATP and recognizing signal peptides which are the earmark of secretory proteins. In contrast, SecB is utilized by only a subset of preproteins to prevent their premature folding and chaperone them to membrane-bound SecA. Using purified components and synthetic signal peptides, we have studied the interaction of SecB with SecA and with SecA-signal peptide complexes in vitro. Using a chemical cross-linking approach, we find that the formation of SecA-SecB complexes is accompanied by a decrease in the level of cross-linking of SecA dimers, suggesting that SecB induces a conformational change in SecA. Furthermore, functional signal peptides, but not dysfunctional ones, promote the formation of SecA-SecB complexes. SecB is also shown to directly enhance the ATPase activity of SecA in a concentration-dependent and saturable manner. To determine the biological consequence of this finding, the influence of SecB on the signal peptide-stimulated SecA/lipid ATPase was studied using synthetic peptides of varying hydrophobicity. Interestingly, the presence of SecB can sufficiently boost the response of signal peptides with moderate hydrophobicity such that it is comparable to the activity generated by a more hydrophobic peptide in the absence of SecB. The results suggest that SecB directly enhances the activity of SecA and provide a biochemical basis for the enhanced transport efficiency of preproteins in the presence of SecB in vivo.     

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.