Dynamics of C-phycocyanin in various deuterated trehalose/water environments measured by quasielastic and elastic neutron scattering

The molecular understanding of protein stabilization by the disaccharide trehalose in extreme temperature or hydration conditions is still debated. In the present study, we investigated the role of trehalose on the dynamics of the protein C-phycocyanin (C-PC) by neutron scattering. To single out the motions of C-PC hydrogen (H) atoms in various trehalose/water environments, measurements were performed in deuterated trehalose and heavy water (D2O). We report that trehalose decreases the internal C-PC dynamics, as shown by a reduced diffusion coefficient of protein H atoms. By fitting the Elastic Incoherent Structure Factor—which gives access to the “geometry” of the internal proton motions—with the model of diffusion inside a sphere, we found that the presence of trehalose induces a significantly higher proportion of immobile C-PC hydrogens. We investigated, by elastic neutron scattering, the mean square displacements (MSDs) of deuterated trehalose/D2O-embedded C-PC as a function of temperature in the range of 40–318 K. Between 40 and ∼225 K, harmonic MSDs of C-PC are slightly smaller in samples containing trehalose. Above a transition temperature of ∼225 K, we observed anharmonic motions in all trehalose/water-coated C-PC samples. In the hydrated samples, MSDs are not significantly changed by addition of 15% trehalose but are slightly reduced by 30% trehalose. In opposition, no dynamical transition was detected in dry trehalose-embedded C-PC, whose hydrogen motions remain harmonic up to 318 K. These results suggest that a role of trehalose would be to stabilize proteins by inhibiting some fluctuations at the origin of protein unfolding and denaturation

Stability of food proteins at high pressure conditions

High pressure (HP) is particularly suited to study protein folding/unfolding, revealing subtle structural rearrangements not accessible by other types of denaturation. HP also has many industrial-scale advantages over heat treatments, including “greener” processing and preservation of nutritional values, colors, and flavors of foods. We have combined in situ HP with small-angle (X-ray and neutron) scattering (SAS) and spectrophotometry to follow the structure in solution of proteins of interest for the food industry. SAS is an essential technique for obtaining structural, but low-resolution, information about proteins, when conventional high-resolution structural biology methods are not possible. I will illustrate this approach with two studies on proteins of food interest: (i) bovine β-lactoglobulin (BLG), a whey protein with a high propensity to bind to various bioactive molecules. We probed by SANS1 and absorbance the effects on pressure stability and reversibility of BLG of the binding of retinol (vitamin A), resveratrol (polyphenol), and biliverdin (linear tetrapyrrole chromophore) to different sites on the protein2, 3. (ii) C-phycocyanin (CPC), a phycobiliprotein from cyanobacteria, to which tetrapyrrole chromophores are covalently attached and which can be used as a natural blue dye in the food industry. We studied by SAXS and absorbance HP-induced CPC unfolding and reversibility from two oligomeric states of the protein as a function of pH. Acknowledgements LLB, SOLEIL, and I2BC facilities are acknowledged for beamtime and proteomic expertise. This work was also supported by ANSO Project No. ANSOCR-PP-2021- 01. References 1. Annighöfer B, et al. High pressure cell to investigate protein unfolding up to 600 MPa by small-angle neutron scattering. Rev Sci Instr 2019;90:025106. 2. Minic S, et al. Effect of ligands on HP-induced unfolding and oligomerization of β-lactoglobulin. Biophysical J 2020;119:2262-74. 3. Minic S, et al. Structure of proteins under pressure: covalent binding effects of biliverdin on beta-lactoglobulin. Biophysical J 2022;121:2514-25

Probing the stability of the food colourant R-phycoerythrin from dried Nori flakes

The high content of vitamins, minerals, antioxidants, and proteins makes red algae Porphyra sp. (Nori) superfood with exceptional health-promoting benefits. Its intense colour originates from R-phycoerythrin (R-PE), phycobiliprotein containing covalently attached tetrapyrrole chromophores: red phycoerythrobilin and orange phycourobilin. The present study aims to characterize the stability of R-PE, a natural colourant with a high potential for application in the food, cosmetic, and pharmaceutical industries. We purified R-PE from dried Nori flakes with a high purity ratio (A560 /A280 ≥5). Far-UV CD spectroscopic showed that α-helix is the dominant secondary structure (75%). The thermal unfolding of α-helix revealed two transitions (Tm1 and Tm2 at 56 and 72°C, respectively), ascribed to the different subunits of R-PE. Absorption measurements showed that high pressure (HP) induces dissociation of R-PE into subunits followed by subunit unfolding. Contrary to temperature, HP treatment showed a significant advantage under applied conditions: the protein unfolding is partly reversible, and the R-PE colour bleaching is minimized. Based on the fluorescence quenching approach, R-PE's binding affinities for Cu2+ and Zn2+ ions were 6.27x105 and 1.71x103 M-1, respectively. Absorption and near-UV/VIS CD spectroscopy suggested conformational changes in protein chromophores upon metal ions binding. Far-UV CD spectroscopy did not reveal that metal binding affects R-PE structure. The obtained results give new insights into the stability of R-PE with a good use-value in replacement of toxic synthetic dyes, preservation of R-PE red colour in fortified food and beverages by HP processing, and as a biosensor for Cu2+ in aquatic life systems. Acknowledgments: This study was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Contract number: 451-03-9/2021-14/200168 and the European Commission, under the Horizon2020, FoodEnTwin Project, GA No. 810752

Combined hydrogels of starch and β-lactoglobulin as matrices for the preservation of C-phycocyanin

The color of food products is an important aspect in food industry, and its preservation remains a big challenge. We aim to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae. For this purpose, we incorporated C-PC in combined starch and β-lactoglobulin (BLG) hydrogels by using a high-pressure (HP) process. Indeed, in thermal treatment, the color derived from C-PC is entirely lost. We characterized the obtained HP gels by both rheology and small-angle X-ray scattering (SAXS). Various formulations of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were tested under HP up to 4,500 bar. A good preservation of the C-PC pigment was established by mixing BLG and starch with C-PC at pH 7, with concentrations of 180, 5, and 10 mg/mL, respectively. Identical component concentrations were maintained in the binary systems. Structure of gels was characterized by SAXS providing insight of C-PC interactions with BLG and starch after HP process which leads to the formation of solid gels with larger mesh compared to two-component systems. This results in enhanced mechanical properties, which were determined by amplitude and frequency sweep measurements using a rheometer with applied plane/plane geometry. Therefore, adding starch, even at small concentration, significantly improves gel visual appearance and mechanical properties. Our study reveals that preservation through HP treatment is more effective than high temperature treatment, as visually observed through the sustained color integrity of C-PC blue dye

The use of starch and β-lactoglobulin composite hydrogels as frameworks for preserving c-phycocyanin

Our study aimed to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae due to its importance in the food industry. We incorporated C-PC into hydrogels formed by combining starch and β-lactoglobulin (BLG) using high-pressure (HP) processing to achieve this objective. Notably, thermal treatment resulted in the complete loss of colour derived from C-PC. We performed a comprehensive characterization of the resulting HP gels by rheology measurements, texture profile analysis (TPA), small-angle X-ray scattering (SAXS), and scanning electron microscopy (SEM). Different compositions of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were processed under high-pressure (HP) conditions reaching up to 4,500 bar. The C-PC pigment was effectively preserved by mixing BLG and starch with C-PC at pH 7, maintaining concentrations of 180, 5, and 10 mg/mL, respectively. The same concentrations of components were retained in the binary systems. Rheological properties of the gels were determined using a rheometer with plane/plane geometry, and texture analysis was conducted through TPA. These findings enabled the assessment of food gel's properties, such as hardness, springiness, chewiness, and cohesiveness. The structural characteristics of the gels were determined by SAXS, offering insights into the interactions between C-PC, BLG, and starch after HP processing. Adding CPC and starch formed solid gels with a larger mesh than the pure BLG gels. SEM scans of the gel surface revealed that all components influenced the overall morphology of gels. Even at low concentrations, the addition of starch notably influenced the gels' visual appearance and mechanical properties. Our investigation highlights the superior effectiveness of HP treatment in the preservation of C-PC compared to high-temperature treatment, evident in the sustained colour integrity of the C-PC blue dye

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics

Molecular mechanisms of peritoneal permeability: interaction between nitric oxide and water channel Aquaporin-1

Thèse de doctorat en sciences biomédicales (physiologie, biologie cellulaire et moléculaire) (SBIM 3)--UCL, 200

An introduction to neutrons for biology

International audienceThe overlap of biology and neutron scattering remains a relatively narrow domain of research. This is partly due to the a priori maladjustment between real space problems and methods based on spatial and temporal correlations. In addition, some major assets of neutron scattering, such as isotopic substitution, can be tricky with biological molecules. More generally, a mutual lack of knowledge of the two concerned communities precluded potential rich interactions in early times. However, the situation changed to the point that, today, biology represents a substantial part of the research activity at neutron facilities. The purpose of this introduction is not to present one more overview of the subject of "neutron scattering" (excellent comprehensive articles are easily accessible to the interested readers [1-4]), but rather to facilitate the reading of the present book by introducing a few neutron scattering notions that may be useful for the community of biologists eventually less familiar with this technique

Effects of sugars on thermal and high-pressure stability of C-phycocyanin from Arthrospira platensis

C-phycocyanin (C-PC), a blue, light-harvesting protein from Arthrospira platensis, is known for industrial application as a food colourant. However, thermal treatment has detrimental effects on C-PC colour due to sensitivity to high temperatures; therefore, its application in the food industry is limited. Hence, stabilisation of the C-PC structure by adding small, food-derived molecules (e.g. sugars) or applying an alternative approach to thermal treatment, such as high-pressure (HP), may allow broader use of this protein. We aimed to study HP and thermal stability of C-PC in the presence of selected sugars (glucose, fructose and sucrose). Ex-situ absorption spectroscopy showed that 18% of glucose, sucrose and fructose solutions, upon incubation at 65℃, exhibit higher colour preservation (91.4, 52.9 and 52.5%, respectively) in comparison to the control (46.9%). HP treatment of C-PC at 450 MPa in 18% solutions of glucose, sucrose and fructose showed 90.1, 93.2 and 74.2% of residual absorbance, respectively, while the HP treatment of control gives 82.3% of residual absorbance. In situ thermal fluorescence measurements revealed that free C-PC has a melting point (Tm) of 55.4°C. In comparison, glucose and sucrose increase Tm of C-PC to 64.4 and 61.4°C, respectively, while fructose does not significantly influence C-PC melting point. In situ HP fluorescence study confirms the stabilisation effects of sugars: the transition pressure (P1/2) of C-PC (230 MPa) is substantially increased in the presence of glucose (277 MPa), sucrose (304 MPa) and fructose (273 MPa). These results showed that HP treatment has significantly less detrimental effects on C-PC colour stability than thermal treatment, and the overall stability of C-PC is substantially increased in the presence of sugars. In contrast, the sugar type determines the stabilisation effect's extent. Consequently, HP treatment of C-PC-containing food could provide an alternative to thermal processing to avoid losing its vivid blue colour