Alfredo Maria Gravagnuolo

Ultraviolet laser-induced cross-linking in peptides

RATIONALE The aim of this study was to demonstrate, and to characterize by high-resolution mass spectrometry that it is possible to preferentially induce covalent cross-links in peptides by using high-energy femtosecond ultraviolet (UV) laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS Three peptides, xenopsin, angiotensin I, and interleukin, individually or in combination, were exposed to high-energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS High-resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS High-energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

UV laser-induced cross-linking in peptides

RATIONALE The aim of this study was to demonstrate, and to characterize by high-resolution mass spectrometry that it is possible to preferentially induce covalent cross-links in peptides by using high-energy femtosecond ultraviolet (UV) laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS Three peptides, xenopsin, angiotensin I, and interleukin, individually or in combination, were exposed to high-energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS High-resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS High-energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

Hydrophobin-coated plates as matrix-assisted laser desorption/ionization sample support for peptide/protein analysis.

Fungal hydrophobins are amphipathic self-assembling proteins. Vmh2 hydrophobin, prepared from mycelial cultures of the basidiomycete fungus Pleurotus ostreatus, spontaneously forms a stable and homogeneous layer on solid surfaces and is able to strongly absorb proteins even in their active forms. In this work, we have exploited the Vmh2 self-assembled layer as a novel coating of a matrix-assisted laser desorption/ionization (MALDI) steel sample-loading plate. Mixtures of standard proteins, as well as tryptic peptides, in the nanomolar-femtomolar range were analyzed in the presence of salts and denaturants. As evidence on a real complex sample, crude human serum was also analyzed and spectra over a wide mass range were acquired. A comparison of this novel coating method with both standard desalting techniques and recently reported on-plate desalting methods was also performed. The results demonstrate that Vmh2 coating of MALDI plates allows for a very simple and effective desalting method suitable for development of lab-on-a-plate platforms focused on proteomic applications.

Green synthesis of luminescent and defect-free bio-nanosheets of MoS2: interfacing two-dimensional crystals with hydrophobins

Solution processing and biofunctionalization of two-dimensional crystals are pivotal for their (biomedical) applications. Here we interface ultrathin layers of MoS2 with the surface active and self-assembling fungal proteins named Vmh2, which belong to the hydrophobin family. We produce few-layered biofunctionalized MoS2 (bio-MoS2) nanosheets via liquid phase exfoliation in a green solvent, and controlled centrifugation; a low-cost and eco-friendly process. The dispersions are investigated by electrophoretic mobility, atomic force microscopy (AFM), UV-Vis, Raman and photoluminescence (PL) spectroscopy. The nanosheets present a defect-free vibrational spectrum, tunable zeta-potential and their photoluminescence is preserved after non-covalent biofunctionalization making them well suited for various biomedical applications.

Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self-Assemble into Functional Amyloid Fibrils

Hydrophobins are fungal proteins whose functions are mainly based on their capability to self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces (HHI). It is widely accepted that class I hydrophobins form amyloid-like structures, named rodlets, which are hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an overall helical structure. We studied the self-assembly of the Class I hydrophobin Vmh2 from Pleurotus ostreatus in aqueous solutions by dynamic light scattering (DLS), thioflavin T (ThT), fluorescence assay, circular dichroism (CD), cryogenic trasmission electron microscopy (cryo-TEM), and TEM. Vmh2 does not form fibrillar aggregates at HHI. It exhibits spherical and fibrillar assemblies whose ratio depends on the protein concentration when freshly solubilized at pH ≥ 7. Moreover, it spontaneously self-assembles into isolated, micrometer long, and twisted amyloid fibrils, observed for the first time in fungal hydrophobins. This process is promoted by acidic pH, temperature, and Ca(2+) ions. A model of self-assembly into amyloid-like structures has been proposed.

One-pot synthesis of a gold nanoparticle-Vmh2 hydrophobin nanobiocomplex for glucose monitoring

HydrophobinVmh2 is a small amphiphilic protein, which self-assembles on different surfaces and naturally interacts with glucose. Here, we report on the synthesis of a nanobiocomplex made of polyethylene glycol, Vmh2 and gold nanoparticles by a one-step process and on its ability to recognise glucose in an aqueous solution at 0.3-0.6-1.2 mg ml(-1) concentrations. Even though the Vmh2 proteins are intrinsically bonded to the gold core, effective glucose interaction monitoring was demonstrated by using dynamic light scattering, ultraviolet-visible, polarization-modulated infrared reflection-absorption and x-ray photoelectron spectroscopies. Experimental results highlighted an affinity constant of 7.3 ± 0.3 mg ml(-1) between the nanobiosystem and the sugar, and a detection sensitivity of 0.13 ± 0.06 a.u./mg ml(-1).

Marine fungi as source of new hydrophobins

Hydrophobins have been described as the most powerful surface-active proteins known. They are produced by filamentous fungi and exhibit a distinct amphiphilic structure determining their self-assembly at hydrophilic-hydrophobic interfaces and surfactant properties which have been demonstrated to be useful for several biotechnological applications. The marine environment represents a vast natural resource of new molecules produced by organisms growing in various stressful conditions. This study was focused on the screening of 100 marine fungi from Mycoteca Universitatis Taurinensis (MUT) for the identification of new hydrophobins. Four different methods were set up to extract hydrophobins of class I and II, from the mycelium or the culture broth of fungi. Six fungi were selected as the best producers of hydrophobins endowed with different characteristics. Their ability to form stable amphiphilic films and their emulsification capacity in the presence of olive oil was evaluated.

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins

Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

Self‐assembly of two hydrophobins from marine fungi affected by interaction with surfaces

Hydrophobins are amphiphilic fungal proteins endowed with peculiar characteristics, such as a high surface activity and an interface triggered self‐assembly. Several applications of these proteins have been proposed in the food, cosmetics and biomedical fields. Moreover, their use as proteinaceous coatings can be effective for materials and nanomaterials applications. The discovery of novel hydrophobins with diverse properties may be advantageous from both the scientific and industrial points of view. Stressful environmental conditions of fungal growth may induce the production of proteins with peculiar features. Two Class I hydrophobins from fungi isolated from marine environment have been recently purified. Herein, their propensity to aggregate forming nanometric fibrillar structures has been compared, using different techniques, such as circular dichroism, dynamic light scattering and Thioflavin T fluorescence assay. Furthermore, TEM and AFM images indicate that the interaction of these proteins with specific surfaces, are crucial in the formation of amyloid fibrils and in the assembly morphologies. These self‐assembling proteins show promising properties as bio‐coating for different materials via a green process. Biotechnol. Bioeng. 2017;114: 2173–2186.