Assembly of the fungal SC3 hydrophobin into functional amyloid fibrils depends on its concentration and is promoted by cell wall polysaccharides.

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.

Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface.

Hydrophobins are small ( approximately 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily beta-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in alpha-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.

Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry.

A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.

Rapid formation of non-native contacts during the folding of HPr revealed by real-time photo-CIDNP NMR and stopped-flow fluorescence experiments.

We report the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, HPr, in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue. The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues, as (i) the observation of transient histidine photo-CIDNP signals during refolding of three of the mutants (F2W, F29W, and F48W) indicates a strong decrease in tryptophan accessibility to the flavin dye; (ii) iodide quenching experiments show that the quenching of the fluorescence of F48W is less efficient for the species formed during the dead-time of the stopped-flow experiment than for the fully native state; and (iii) kinetic fluorescence anisotropy measurements show that the tryptophan side-chain of F48W has lower mobility in the dead-time intermediate state than in both the fully denatured and fully native states. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues. This process may be important in preventing the protein from aggregating prior to the acquisition of native-like structure in which hydrophobic residues are exposed in order to play their role in the function of the protein. The phenylalanine residue at position 48 is likely to be of particular interest in this regard as it is involved in the binding to enzymes I and II that mediates the transfer of a phosphoryl group between the two enzymes.

Oligomerization of hydrophobin SC3 in solution: from soluble state to self-assembly.

Hydrophobin SC3 is a protein with special self-association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self-association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self-association in water because this is the starting point for the other two association processes. Size-exclusion chromatography was used to fractionate soluble-state SC3. Real-time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl-labeled SC3. Size-exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4 degrees C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short-lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 microg/mL. This value is similar to the critical concentration for SC3 self-assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self-assembly at hydrophobic/hydrophilic interfaces.

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates.

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.

Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-beta-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion.

Use of hydrophobins in formulation of water insoluble drugs for oral administration.

The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6+/-2- and 4+/-3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral formulation by increasing bioavailability of CyA 2.3+/-0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p<0.001 in T(max)) of the drug, with a lower peak concentration (C(max) 1.8 mg ml(-1)) at a later time point (T(max) 9+/-2 h) compared to Neoral (C(max) 2.2 mg ml(-1); T(max) 3.2+/-0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.

Adhesion receptors mediate efficient non-viral gene delivery.

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

A biological porin engineered into a molecular, nanofluidic diode.

We changed the nonrectifying biological porin OmpF into a nanofluidic diode. To that end, we engineered a pore that possesses two spatially separated selectivity filters of opposite charge where either cations or anions accumulate. The observed current inhibition under applied reverse bias voltage reflects, we believe, the creation of a zone depleted of charge carriers, in a sense very similar to what happens at the np junction of a semiconductor device.

Novel surface display system for proteins on non-genetically modified gram-positive bacteria.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization.

The present work reports the use of non-living non-recombinant bacteria as a delivery system for mucosal vaccination. Antigens are bound to the cell-wall of pretreated Lactococcus lactis, designated as Gram-positive enhancer matrix (GEM), by means of a peptidoglycan binding domain. The influence of the GEM particles on the antigen-specific serum antibody response was studied. Following nasal immunization with the GEM-based vaccines, antibody responses were induced at systemic and local levels. Furthermore, different GEM-based vaccines could be used consecutively in the same mice without adverse effects or loss of activity. Taken together, the results evidence the adjuvant properties of the GEM particles and indicate that GEM-based vaccines can be used repeatedly and are particularly suitable for nasal immunization purposes.

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria.

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.

The smallest resonance energy transfer acceptor for tryptophan.

The utility of diazirine ligands as acceptors in resonance energy transfer (RET) distance measurements with tryptophan or tryptophan analogues as donor is reported. The principle is demonstrated for a diazirine derivative of d-mannitol, 2-azi-2-deoxy-d-arabino-hexitol, and single-tryptophan-containing mutants of the mannitol transporter, EIImtl, from E. coli. The Förster distance is 10 A for the tryptophan-diazirine donor-acceptor couple, allowing the measurement of distances up to 17 A. The versatility of tryptophan as an intrinsic spectroscopic probe in protein chemistry and the small size of the diazirine group makes this a very attractive donor-acceptor couple for accurate RET distance information in protein chemistry.

Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. II. Hydrodynamic studies.

This study describes the hydrodynamic properties of the repetitive domain of high molecular weight (HMW) wheat proteins, which complement the small-angle scattering (SANS) experiments performed in the first paper of this series. The sedimentation coefficients, s(0), and diffusion coefficients, D(0), were obtained from the homologous HMW proteins dB1 and dB4 that were cloned from the gluten protein HMW Dx5, and expressed in Escherichia coli. Monodisperse conditions for accurate determination of s(0) and D(0), were obtained by screening a series of buffers using dynamic light scattering. For the first time, hydrodynamic parameters were obtained on monodisperse samples that enabled the determination of the monomeric size and shape. The hydrodynamic values determined on dB1 and dB4 were used to test the worm-like chain (WLC) model that was proposed in the SANS studies. The successful matching of two separately obtained hydrodynamic parameters of dB1 and dB4 using the WLC model provides further evidence for the WLC model. The small discrepancy between the hydrodynamic and scattering data, possibly coming from the excluded volume effect, was compensated by a solvation layer of 1-2 water molecules thick around the protein in the WLC model. The solvation of the central domain is much higher than those of the terminal domains of the HMW subunits. This difference emphasizes the dual role of HMW wheat gluten proteins in water-binding and aggregation.

Molecular dynamics study of the folding of hydrophobin SC3 at a hydrophilic/hydrophobic interface.

Hydrophobins are fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes. These assemblages are extremely stable and posses the remarkable ability to invert the polarity of the surface on which they are adsorbed. Neither the three-dimensional structure of a hydrophobin nor the mechanism by which they function is known. Nevertheless, there are experimental indications that the self-assembled form of the hydrophobins SC3 and EAS at a water/air interface is rich with beta-sheet secondary structure. In this paper we report results from molecular dynamics simulations, showing that fully extended SC3 undergoes fast (approximately 100 ns) folding at a water/hexane interface to an elongated planar structure with extensive beta-sheet secondary elements. Simulations in each of the bulk solvents result in a mainly unstructured globular protein. The dramatic enhancement in secondary structure, whether kinetic or thermodynamic in origin, highlights the role interfaces between phases with large differences in polarity can have on folding. The partitioning of the residue side-chains to one of the two phases can serve as a strong driving force to initiate secondary structure formation. The interactions of the side-chains with the environment at an interface can also stabilize configurations that otherwise would not occur in a homogenous solution.

Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. I. Small-angle neutron scattering.

The solution structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins has been investigated for a range of concentrations and temperatures using mainly small-angle neutron scattering. A representative part of the repetitive domain (dB1) was studied as well as an "oligomer" basically consisting of four dB1 units, which has a length similar to the complete central domain. The scattering data over the entire angular range of both proteins are in quantitative agreement with a structural model based on a worm-like chain, a model frequently used in polymer theory. This model describes the "supersecondary structure" of dB1 and dB4 as a semiflexible cylinder with a length of about 235 and 900 A, respectively, and a cross-sectional diameter of about 15 A. The flexibility of both proteins is characterized by a persistence length of about 13 A. Their structures are thus quantitatively identical, which implies that the central HMW domain can be elongated while retaining its structural characteristics. It seems conceivable that the flexible cylinder results from a helical structure, which resembles the beta-spiral observed in earlier studies on gluten proteins and elastin. However, compared to the previously proposed structure of a (stiff) rod, our experiments clearly indicate flexibility of the cylinder.

Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking.

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.

Characterization of single-tryptophan mutants of histidine-containing phosphocarrier protein: evidence for local rearrangements during folding from high concentrations of denaturant.

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.