Francesco Milano

Photosynthetic Machineries in Nano-Systems

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Reversible Binding of Metal Ions onto Bacterial Layers Revealed by Protonation-Induced ATR-FTIR Difference Spectroscopy

The ability of microorganisms to adhere to abiotic surfaces and the potentialities of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy have been exploited to study protonation and heavy metal binding events onto bacterial surfaces. This work represents the first attempt to apply on bacteria the recently developed method known as perfusion-induced ATR-FTIR difference spectroscopy. Such a technique allows measurement of even slight changes in the infrared spectrum of the sample, deposited as a thin layer on an ATR crystal, while an aqueous solution is perfused over its surface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surface of Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni(2+) with surface protonable groups of this microorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons. Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterial spectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected reveal that carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni(2+), establishing with this ion relatively weak coordinative bonds.

Enhancing the Light Harvesting Capability of a Photosynthetic Reaction Center by a Tailored Molecular Fluorophore

Light machine: The simplest photosynthetic protein able to convert sunlight into other energy forms is covalently functionalized with a tailored organic dye to obtain a fully functional hybrid complex that outperforms the natural system in light harvesting and conversion ability.

Bioconjugation of hydrogen-bonded organic semiconductors with functional proteins

We demonstrate the direct bioconjugation of hydrogen-bonded organic semiconductors with two different complex functional proteins in an aqueous environment. The representative semiconductors are epindolidione and quinacridone, materials used in devices in the form of vacuum-evaporated polycrystalline films. First, these molecules in thin films react spontaneously with N-hydroxysuccinimide functionalized linkers: disuccinimidyl suberate and succinimidyl biotinate. The suberate linker is then used to covalently bind the Rhodobacter sphaeroides reaction centre (RC), the key photoenzyme for conversion of light into electrical charges in photosynthetic bacteria. Similarly, the biotin linker is used to bridge streptavidin to the surface of the hydrogen-bonded semiconductor film. Multiple-reflection infrared spectroscopy, water contact angle measurements, and atomic force microscopy are used to verify surface functionalization. The presence and functional integrity of the immobilized proteins are demonstrated by specific experiments: a charge recombination kinetics assay in the case of the RC, and photoluminescence measurements for quantum dot-labelled streptavidin. As key results of our work, we have shown that upon bioconjugation, the semiconductors preserve their favourable electrical properties: as evidenced by photoconductor devices operating under water sensitized by the RC, and thin film transistor measurements before and after bioconjugation. These are enabling steps for using hydrogen-bonded semiconductors as platforms for multifunctional bioelectronics devices.

Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg(2+)) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg(2+) is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Response of membrane protein to the environment: the case of photosynthetic Reaction Centre

The role of the environment on the function and structure of enzymes is discussed in the case of the photosynthetic Reaction Centre (RC) reconstituted in proteoliposomes. The reconstitution procedure is illustrated in detail and the effect of the bilayer upon the activity of the enzyme is discussed. The combined use of ENDOR spectroscopy and flash photolysis clearly demonstrate that the enzyme isolation procedure induces some structural changes, relative to the native state, that are completely reversible once the protein is reconstituted in the phospholipid bilayer.

Trapping of a long-living charge separated state of photosynthetic reaction centers in proteoliposomes of negatively charged phospholipids

AbstractReaction centers from the purple bacterium Rhodobacter sphaeroides strain R-26.1 were purified and reconstituted in proteoliposomes formed by the anionic phospholipids phosphatidylglycerol, phosphatidylserine and phosphatidylinositol and by the zwitterionic phospholipid phosphatidylcholine by size-exclusion chromatography in the dark and under illumination. We report the large stabilizing effect induced by anionic phospholipids on the protein charge separated state which results trapped in a long-living (up to tens of minutes) state with a yield up to 80%. This fully reversible state is formed in oxygenic conditions regardless the presence of the secondary quinone QB and its lifetime and relative yield increase at low pH. In proteoliposomes formed with QA-depleted reaction centers (RCs) the resulting protein is very light-sensitive and the long living charge separated state is not observed. The data collected in negatively charged proteoliposomes are discussed in terms of the electrostatic effect on the primary quinone acceptor and compared with similar long living species reported in literature and obtained in anionic, zwitterionic, and non-ionic detergents.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells

Significance The photosynthetic reaction center (RC), an integral membrane protein at the core of bioenergetics of all autotrophic organisms, has been reconstituted in the membrane of giant unilamellar vesicles (RC@GUV) by retaining the physiological orientation at a very high percentage (90 ± 1%). Owing to this uniform orientation, it has been possible to demonstrate that, under red-light illumination, photosynthetic RCs operate as nanoscopic machines that convert light energy into chemical energy, in the form of a proton gradient across the vesicle membrane. This result is of great relevance in the field of synthetic cell construction, proving that such systems can easily transduce light energy into chemical energy eventually exploitable for the synthesis of ATP. Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min−1. Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

''Garnishing'' the photosynthetic bacterial reaction center for bioelectronics

The photosynthetic reaction center is an extraordinarily efficient natural photoconverter, which can be ideally used in combination with conducting or semiconducting interfaces to produce electrical signals in response to absorption of photons. The actual applicability of this protein in bioelectronic devices critically depends on the finding of (a) suitable deposition methods enabling controlled addressing and precise orientation of the protein on electrode interfaces and (b) chemical manipulation protocols able to tune and enhance protein light absorption in specific or broader spectral regions. Literature reports several examples of approaches to fulfill these requirements, which have faced in different ways the fundamental issues of assembling the biological component and non-natural systems, such as electrode surfaces and artificial light harvesting components. Here we present a short overview of the main methods reported to accomplish both the objectives by properly “garnishing” the photosynthetic reaction center (RC) via chemical modifications.

Hybrid assemblies of fluorescent nanocrystals and membrane proteins in liposomes.

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.