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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.