Exploring hydrodynamic cavitation for citrus waste valorisation in Malta: from beverage enhancement to potato sprouting suppression and water remediation.

Introduction: The endorsement of circular economy, zero-waste, and sustainable development by the EU and UN has promoted non-thermal technologies in agro-food and health industries. While northern European countries rapidly integrate these technologies, their implementation in Mediterranean food-supply chains remains uncertain. Aims: We evaluated the usefulness of hydrodynamic cavitation (HC) for valorizing orange peel waste in the fresh orange juice supply chain of the Maltese Islands. Method: We assessed: a) the effectiveness of HC in extracting bioactive compounds from orange peels (Citrus sinensis) in water (35°C) and 70% (v/v) ethanol (-10°C) over time, compared to conventional maceration, and b) the potato sprouting-suppression and biosorbent potential of the processed peel for copper, nitrate, and nitrite binding. Results: Prolonged HC-assisted extractions in water (high cavitation numbers), damaged and/or oxidized bioactive compounds, with flavonoids and ascorbic acid being more sensitive, whereas cold ethanolic extractions preserved the compounds involved in radical scavenging. HC-processing adequately modified the peel, enabling its use as a potato suppressant and biosorbent for copper, nitrate, and nitrite. Conclusion: Coupling HC-assisted bioactive compound extractions with using leftover peel for potato-sprouting prevention and as biosorbent for water pollutant removal offers a straightforward approach to promoting circular economic practices and sustainable agriculture in Malta.

Improved fluorescent phytochromes for in situ imaging.

Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells.

The multiple assemblies of VDAC: from conformational heterogeneity to ß-aggregation and amyloid formation.

From their cellular localisation, to their atomic structure and their involvement in mitochondrial-driven cell death, voltage-dependent anion channels (VDACs) have challenged the scientific community with enigmas and paradoxes for over four decades. VDACs form active monomer channels in lipid bilayers, but they can also organise in multimeric assemblies. What induces, regulates and/or controls the monomer-multimer dynamics at the cellular level is not known. However, these state transitions appear to be relevant for mitochondria in making life or death decisions and for driving developmental processes. This review starts with a general introduction on VDACs and continues by examining VDAC oligomerisation/aggregation in light of recent discussions on VDAC-ß-amyloid interactions and their involvement in Alzheimer's disease.

Voltage-dependent anion channels: the wizard of the mitochondrial outer membrane.

Voltage dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane. Although they are essential in metabolite exchange, cell defense and apoptosis, the molecular mechanism of these VDAC-mediated processes remains elusive. Here we review recent progress in terms of VDACs' structure and regulation, with a special focus on the molecular aspects of gating and the interaction with effector proteins.

Structure-based engineering of a minimal porin reveals loop-independent channel closure.

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded ß-barrel.

Tyrosine 263 in cyanobacterial phytochrome Cph1 optimizes photochemistry at the prelumi-R→lumi-R step.

We report a low-temperature fluorescence spectroscopy study of the PAS-GAF-PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi-R photoreaction (2.5-3.1 kJ mol(-1)) and fluorescence quantum yields (0.29-0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (ΦPr→Pfr) was observed primarily at the prelumi-R S0 bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi-R or Pr, lowering ΦPr→Pfr from 0.13 in the wild type to 0.05-0.07 in the mutants. We suggest that the Ea activation barrier in the Pr* S1 excited state might correspond to the D-ring (C19) carbonyl - H290 hydrogen bond or possibly to the hindrance caused by the C13(1) /C17(1) methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi-R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi-R for subsequent rearrangement processes culminating in Pfr formation.

The D-ring, not the A-ring, rotates in Synechococcus OS-B' phytochrome.

Phytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250-254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B'. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B' phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family.

Flexibility of the N-terminal mVDAC1 segment controls the channel's gating behavior.

Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel's gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an "asymmetric" gating behavior, with closure only at one electric field's orientation depending on the channel's orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel's gating behavior.

On the collective nature of phytochrome photoactivation.

The red/far-red-sensing biological photoreceptor phytochrome is a paradigmatic two-state signaling system. The two thermally stable states are interconverted via a photoreaction of the covalently bound tetrapyrrole chromophore. Applying recently developed solid-state nuclear magnetic resonance, we study both the chromophore and its protein pocket in the Pr (red-absorbing) and Pfr (far-red-absorbing) states. The observations show that the phototransformation combines local chemical reactions with a mesoscopic transition of order. Both the chromophore and its binding pocket are quasi-liquid and disordered in Pr, yet quasi-solid and ordered in Pfr. Possible biochemical implications are discussed.

Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1.

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.

Signaling kinetics of cyanobacterial phytochrome Cph1, a light regulated histidine kinase.

Cyanobacterial phytochrome 1 (Cph1) is a red/far-red light regulated histidine kinase, which together with its response regulator (Rcp1) forms a two-component light signaling system in Synechocystis 6803. In the present study we followed the in vitro autophosphorylation of Cph1 and the subsequent phosphotransfer to Rcp1 in different ionic milieus and following different light treatments. Both processes were red/far-red reversible with activity manifested in the Pr ground state (in darkness or after far-red irradiation) and with strongest activities being exhibited in the presence of Mn(2+). In vivo and in vitro assembled holoproteins in the Pr state displayed at least 4-fold higher efficiencies (k(cat)/K(m)) for autophosphorylation and phosphotransfer than the apoprotein or the holoprotein at photoequilibrium in red light. The reduced activities observed following red light treatments were consistent with the Pfr state being enzymatically inactive. Thus, both the rate of kinase autophosphorylation and the rate of phosphotransfer regulate the phosphorylation state of the response regulator, consistent with the rotary switch model regulating accessibility of the histidine target.

Two ground state isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes in a canonical phytochrome.

Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using (1)H-(13)C magic-angle spinning NMR spectroscopy of the sensor module of cyanobacterial phytochrome Cph1. Two isoforms of the red-light absorbing Pr ground state are identified. Conclusive evidence that photoisomerization occurs at the C15-methine bridge leading to a ß-facial disposition of the ring D is presented. In the far-red-light absorbing Pfr state, strong hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 hold the chromophore in a tensed conformation. Signaling is triggered when Asp-207 is released from its salt bridge to Arg-472, probably inducing conformational changes in the tongue region. A second signal route is initiated by partner swapping of the B-ring propionate between Arg-254 and Arg-222.

NMR spectroscopic investigation of mobility and hydrogen bonding of the chromophore in the binding pocket of phytochrome proteins.

For a complete understanding of the light reception of phytochrome proteins, a detailed study of the structure and dynamics of the binding pocket at atomic resolution is required. Structures from X-ray crystallography and NMR spectroscopy are available and have been able to provide a picture of the binding pocket. NMR spectroscopy has, in addition, shown that the chromophore exhibits noticeable dynamics in the binding pocket of the cyanobacterial phytochrome Cph1. Herein, NMR spectroscopy is used to investigate further the mobility of the chromophore by analyzing the line widths of the resonances of the chromophore in various environments, in particular other protein environments. It is shown that the chromophore exhibits a different mobility in the binding pocket of the bacterial phytochrome Agp1 than in that of the cyanobacterial phytochrome Cph1. Finally, it is shown that NMR spectroscopy is capable of detecting hydrogen bonds in the binding pocket of phytochromes by observing slow exchange of protons in the amino acid side chains.

Dwelling in the dark: procedures for the crystallography of phytochromes and other photochromic proteins.

Crystallization of phytochromes and other photochromic proteins is hampered by the conformational changes that they undergo on exposure to light. As a canonical phytochrome, cyanobacterial Cph1 switches between two stable states upon absorption of red/far-red light. Consequently, it is mandatory to work in darkness from protein purification to crystal cryoprotection in order to ensure complete occupancy of one state or the other. With the simple and inexpensive methods that have been developed, phytochromes and other photochromic molecules can effectively be handled and crystallized, as has been demonstrated by the solution of the three-dimensional structure of the Cph1 sensory module.

AcrB et al.: Obstinate contaminants in a picogram scale. One more bottleneck in the membrane protein structure pipeline.

Heterologous expression of integral membrane proteins from Helicobacter pylori 26695 in Escherichia coli enabled the identification of 17 candidates for purification and subsequent crystallization. 45% of the purified proteins were contaminated with what was later identified as the multidrug efflux pump (AcrB)of E. coli, and 17% with the succinate dehydrogenase. While additional purification steps ensured removal of succinate dehydrogenase, they failed to remove AcrB completely, leaving picogram amounts present infractions intended for 3D-crystallization. Two of these targets, the Na+ dependent D-glucose/D-galactose transporter (GluP-HP1174) and the carbon starvation protein A (CstA-HP1168), produced small crystals(<40 lm). Crystals from the GluP preparation diffracted to 4.2 A resolution and belonged to the rhombohedral space group H32. Subsequent molecular replacement proved that these crystals were derived from a contaminant, the efflux transporter AcrB. This unexpected crystallization of AcrB from picogram amounts was observed in six new conditions. The systematic occurrence of AcrB in membrane preparations stems from the upregulation of its transcription in response to the stress induced by the expression of a selected target. This, along with its tendency to crystallize in the picogram scale, poses a serious concern in membrane protein expression using heterologous hosts harbouring AcrB.

The sodium-dependent D-glucose transport protein of Helicobacter pylori.

Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.

Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR.

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of (13)C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly (13)C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.

Expression screening of integral membrane proteins from Helicobacter pylori 26695.

The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins < or =40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.

Collection and characterisation of bacterial membrane proteins.

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.