George T. Robillard

Structural Characterization of the Hydrophobin SC3, as a Monomer and after Self-Assembly at Hydrophobic/Hydrophilic Interfaces

Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

ABSTRACT 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.

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 α‐helical state that is formed upon binding to a hydrophobic solid, and the β‐sheet state, which is formed at the air–water interface. Here, we show that the formation of the β‐sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the α‐helical state of SC3. The second intermediate is rich in β‐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.

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°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 μg/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.

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.

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Abstract Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time. The three-dimensional structure of yeast tRNAPhe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.

A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [32P]phosphoenolpyruvate from [gamma-32P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of 32P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.

Multiple phosphorylation events regulate the activity of the mannitol transcriptional regulator MtlR of the Bacillus stearothermophilus phosphoenolpyruvate-dependent mannitol phosphotransferase system

d-Mannitol is taken up byBacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICBmtl. The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICBmtl. An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICBmtl and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICBmtl via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [3H]mannitol phosphate in the absence of IIAmtl. Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlR-MtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICBmtl have not been described before; they could also play a role in other PRD-containing proteins.

The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system

The region of the surface of the histidine‐containing protein (HPr) which interacts with the A domain of the mannitol‐specific Enzyme II (IIAmtl) has been mapped by titrating the A‐domain into a solution of 15N‐labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty‐five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of IIAmtl while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of IIAmtl resulted in chemical shift changes in a sub‐set of the above residues; these were located more in the vicinity of the active site phospho‐histidine. Phosphorylation of the HPr/IIAmtl complex resulted in a HSQC spectrum which was indistinguishable from the P‐HPr spectrum in the absence of IIAmtl indicating that, as expected, the complex P‐HPr/P‐IIAmtl does not exist even at the high concentrations necessary for NMR.

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.