129 research outputs found
Retardation of Protein Dynamics by Trehalose in Dehydrated Systems of Photosynthetic Reaction Centers. Insights from Electron Transfer and Thermal Denaturation Kinetics
No full textConformational protein dynamics is known to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presence of glass forming disaccharides, like trehalose, resulting in enhanced protein thermal stability. To shed light on such matrix effects, we have compared the retardation of protein dynamics in photo-synthetic bacterial reaction centers (RC) dehydrated at controlled relative humidity in the absence (RC films) or in the presence of trehalose (RC-trehalose glasses). Small scale RC dynamics, associated with the relaxation from the dark-adapted to the light-adapted conformation, have been probed up to the second time scale by analyzing the kinetics of electron transfer from the photoreduced quinone acceptor (Q(A)(-)) to the photoxidized primary donor (P+) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s. A more severe inhibition of dynamics is found in RC trehalose glasses than in RC films: only in the latter system does a complete relaxation to the light-adapted conformation occur even at extreme dehydration, although strongly retarded. To gain insight into the large scale RC dynamics up to the time scale of days, the kinetics of thermal denaturation have been studied at 44 degrees C by spectral analysis of the Q(x) and Q(y) bands of the RC bacteriochlorin cofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 10(4). Upon increasing m, denaturation is slowed progressively, and above m similar to 500 the RC is stable at least for several days. The stronger retardation of RC relaxation and dynamics induced by trehalose is discussed in the light of a recent molecular dynamics simulation study performed in matrixes of the model protein lysozyme with and without trehalose. We suggest that the efficiency of trehalose in retarding RC dynamics and preventing thermal denaturation stems mainly from its propensity to form and stabilize extended networks of hydrogen bonds involving sugar, residual water, and surface residues of the RC complex and from its ability of reducing the free volume fraction of protein alone matrixes
Ionic liquids effects on the permeability of photosynthetic membranes probed by the electrochromic shift of endogenous carotenoids
No full textAbstractIonic liquids (ILs) are promising materials exploited as solvents and media in many innovative applications, some already used at the industrial scale. The chemical structure and physicochemical properties of ILs can differ significantly according to the specific applications for which they have been synthesized. As a consequence, their interaction with biological entities and toxicity can vary substantially. To select highly effective and minimally harmful ILs, these properties need to be investigated. Here we use the so called chromatophores – protein-phospholipid membrane vesicles obtained from the photosynthetic bacterium Rhodobacter sphaeroides– to assess the effects of imidazolinium and pyrrolidinium ILs, with chloride or dicyanamide as counter anions, on the ionic permeability of a native biological membrane. The extent and modalities by which these ILs affect the ionic conductivity can be studied in chromatophores by analyzing the electrochromic response of endogenous carotenoids, acting as an intramembrane voltmeter at the molecular level. We show that chromatophores represent an in vitro experimental model suitable to probe permeability changes induced in cell membranes by ILs differing in chemical nature, degree of oxygenation of the cationic moiety and counter anion
Effect of Dehydration on Light-Adapted States of Bacterial Reaction Centers Studied by Time-Resolved Rapid-Scan FTIR Difference Spectroscopy
No full textDehydration is known to affect the rate of electron transfer backreaction from the light-induced charge separation state P+QA− to the neutral ground state PQA in photosynthetic bacterial Reaction Centers. On the other hand, a 20 s continuous illumination period has been demonstrated to induce (at 297 K) formation of one or more light-adapted states at different levels of dehydration; these light-adapted states are believed to be related to peculiar response(s) from the protein. In this work, we applied time-resolved rapid-scan FTIR difference spectroscopy to investigate the protein response under dehydrated conditions (RH = 11%) at 281 K both after a flash and under prolonged continuous illumination. Time-resolved FTIR difference spectra recorded after a laser flash show a protein recovery almost synchronous to the electron transfer backreaction P+QA− → PQA. Time-resolved FTIR difference spectra recorded after 20.5 s of continuous illumination (RH = 11%, T = 281 K) surprisingly show almost the same kinetics of electron transfer back reaction compared to spectra recorded after a laser flash. This means that the mechanism of formation of a light-adapted stabilized state is less effective compared to the same hydration level at 297 K and to the RH = 76% hydration level (both at 281 K and 297 K). Time-resolved FTIR difference spectra after continuous illumination also suggest that the 1666 cm−1 protein backbone band decays faster than marker bands for the electron transfer back reaction P+QA− → PQA. Finally, FTIR double-difference spectra (FTIR difference spectrum recorded after 18.4 s illumination minus flash-induced FTIR difference spectrum) suggest that at RH = 11%, a light-adapted state different from the one observed at RH = 76% is formed. A possible interpretation is that at RH = 11%, the protein response is modified by the fact that only protons can move easily, differently from water molecules, as instead observed for RH = 76%. This probably makes the formation of a real light-adapted P+QA− stabilized state at RH = 11% unfeasible
La strategia dei viventi per la sopravvivenza in assenza di acqua
No full textSi esamina il fenomeno dell'anidrobiosi, tracciando lo sviluppo storico delle conoscenze dai contributi di Spallanzani ad oggi. E' posto in particolare rilievo come lo studio della dinamica interna delle macromolecole e dell'interazione col solvente abbia contribuito ad una analisi del fenomeno a livello molecolare
Print-Light-Synthesis of ruthenium oxide thin film electrodes for electrochemical sensing applications
No full textPrint-Light-Synthesis (PLS) combines the inkjet printing of a ruthenium precursor ink with the simultaneous photo-induced generation of ruthenium oxide films. During PLS, inkjet-printing generates on conductive as well as insulating substrates micrometer-thin reaction volumes that contain with high precision defined precursor loadings. Upon direct UV light irradiation, the Ru precursor converts to RuO 2 while all other ink components escape in the gas phase. No post PLS processes are required, and the as-obtained RuO 2 films can be immediately used as electrochemical devices. Two-dimensional RuO 2 patterns with micrometric resolution and highlycontrolled ruthenium loadings (few µg/cm 2) are realized. Thin RuO 2 films are generated on insulating substrates, such as polyimide, as well as individual RuO 2 particles on conductive substrates, such as graphene layers. The RuO 2 films are characterized by electron microscopy and spectroscopic techniques. The sensoristic applicability of the PLS-RuO 2 electrodes is demonstrated by potentiometric pH sensing in cell cultures and amperometric detection of L-cysteine. For pH sensing the RuO 2 film electrodes show Nernstian sensitivity. L-cysteine detection of RuO 2-modified graphene electrodes showed an electrocatalytical effect and resulted in the possibility of selectively detecting L-Cysteine also in presence of the interfering compound uric acid. ☆ This article is part of a special issue entitled: '11th SMCBS 2023 Workshop' published in Bioelectrochemistry.GR-LU
The 2M1 dioctahedral mica polytype: a crystal chemical study
No full textThe structure of dioctahedral true micas such as muscovite and celadonitic muscovite (2M1 polytype, Space Group C2/c) is mostly affected by variations of the octahedral Al content ([vi]Al). Crystals with greater Mg, Fe substitutions (i.e., celadonitic muscovite) reduce the dimensional difference between the larger trans-oriented M(1) site and smaller cis-oriented M(2) octahedral site. The octahedral anionic position O(4) is displaced from the center of the hexagon, defined by O(31) and O(32) oxygen atoms (i.e., “octahedral hexagon”), both on and off the (001) plane. The distance between interlayer cation A and O(4) is smaller in more substituted species, thus providing different orientations of the O(4)-H vector, as a function of [vi]Al. Octahedral distances and are expressed as a function of cell parameters and [vi]Al content, thus allowing an approximate estimate of site dimensions. These approximations are useful when a detailed structural refinement is not available. In celadonitic muscovite, the octahedral hexagon mean edge is not significantly affected by [vi]Al content. The [vi]Al increase produces both a decrease in cell lateral dimensions and a distorted “octahedral hexagon”. The decrease in a and b is consistent with a decrease of , whereas the distortion of the “octahedral hexagon” is consistent with an increase of , because an irregular hexagon produces a longer mean edge than a regular hexagon of equal area.The tetrahedral mean basal edge is reduced as celadonitic substitution progresses. The tetrahedral rotation angle was thus found to increase from celadonite to muscovite. However in muscovite with [vi]Al content between 1.8 and 2.0 atoms per formula unit (apfu), approaches a saturation value, thus showing a proportional increase of tetrahedral and octahedral sheet lateral dimensions. Furthermore, alpha variation allows a coarse approximation of the threshold [vi]Al content, below which celadonitic substitution may not progress
Composizione, microstruttura, e morfologie dei perni
No full textI capitolo descrive la composizione, microstruttura e la morfologia dei perni disponibili sul mercato
Protein Immobilization Capabilities of Sucrose and Trehalose Glasses: The Effect of Protein/Sugar Concentration Unraveled by High-Field EPR
No full textDisaccharide glasses
are increasingly used to immobilize proteins
at room temperature for structural/functional studies and long-term
preservation. To unravel the molecular basis of protein immobilization,
we studied the effect of sugar/protein concentration ratios in trehalose
or sucrose matrixes, in which the bacterial photosynthetic reaction
center (RC) was embedded as a model protein. The structural, dynamical,
and H-bonding characteristics of the sugar–protein systems
were probed by high-field W-band EPR of a matrix-dissolved nitroxide
radical. We discovered that RC immobilization and thermal stabilization,
being independent of the protein concentration in trehalose, occur
in sucrose only at sufficiently low sugar/protein ratios. EPR reveals
that only under such conditions does sucrose form a microscopically
homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe.
We conclude that the protein immobilization capability depends critically
on the propensity of the glass-forming sugar to create intermolecular
H-bond networks, thus establishing long-range, homogeneous connectivity
within the matrix
Coupling between Electron Transfer and Protein-Solvent Dynamics: FTIR and Laser-Flash Spectroscopy Studies in Photosynthetic Reaction Center Films at Different Hydration Levels
No full textWe report on the relationship between electron transfer, conformational dynamics, and hydration in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides. The kinetics of electron transfer from the photoreduced quinone acceptor (Q A -) to the photo-oxidized primary donor (P +), a charge recombination process sensitive to the conformational dynamics of the RC, has been analyzed at room temperature in dehydrated RC-detergent films as a function of the residual water content under controlled relative humidity (r). The hydration level was evaluated by FTIR spectroscopy from the area of the combination band of water (5155 cm -1). Sorption isotherms fit the Hailwood and Horrobin model and indicate a significant contribution to hydration of the detergent belt surrounding the RC. Spectral analysis of the water combination and association (2130 cm -1) bands suggests strong rearrangements in the hydrogen-bonding organization upon depletion of the hydration shell of the complex. In parallel with these changes, following dehydration below a critical threshold (r ≃ 40%), the kinetics of P +Q A - recombination become progressively faster and distributed in rate. When r is decreased from 40% to 10% the average rate constant 〈k〈 increases from 15 to 40 s -1, mimicking the behavior of the hydrated system at cryogenic temperatures. We infer that extensive dehydration inhibits dramatically the relaxation from the dark- to the light-adapted conformation of the RC as well as interconversion among lower tier conformational substates. The RC dynamics probed by P +Q A - recombination appear therefore controlled by the thermal fluctuations of the hydration shell. At r < 10% an additional, much faster (〈k〈 ≃ 3000 s -1) kinetic phase of P +Q A - recombination is observed. We suggest such a fast recombination arises from removal of a pool of RC-bound water molecules which are essential to stabilize the primary charge-separated state at physiological conditions
Exploring the coupling between electron transfer and protein dynamics in photosynthetic reaction centers embedded into dehydrated amorphous matrices
No full textThe interplay between protein dynamics and electron transfer (ET)
has been extensively investigated in the bacterial photosynthetic reaction
center (RC) from Rhodobacter sphaeroides by hampering RC internal
motions at low temperatures [1]. Alternatively, the RC dynamics can
be inhibited at room temperature by incorporating the RC into
dehydrated trehalose matrices [2]. In the glasses the recombination
kinetics of the charge separated state P+QA
are accelerated and
distributed in rate as compared to solution, mimicking at room
temperature the effects observed at 10 K in water-glycerol. This is
taken to indicate inhibition of the RC relaxation from the dark- to the
light-adapted conformation, as well as of the RC thermal fluctuations
[1,2]. We proposed that the inhibition is mediated by residual water
molecules of the RC hydration shell which bridge protein surface
groups with trehalose molecules of the matrix by forming a network
of multiple hydrogen bonds [3]. Consistently, similar effects have
been observed also in RC films dehydrated in the absence of sugar [4].
However, striking differences are found between RC-trehalose glasses
and RC-films: (a) The thermal stability of the RC is tremendously
enhanced in the trehalose matrix. (b) In RC-trehalose matrices, the
P+QA
recombination after a few seconds of continuous photoexcitation
is only partially decelerated as compared to the one recorded
after a laser flash, whereas in dried RC films a comparable period of
continuous illumination leads to a total recovery of the kinetics
observed in the hydrated system. These data indicate that in films the
protein dynamics can be easily regained, in contrast to trehalose
glasses, which reveal much stronger structural constraints. We are
extending these studies to a series of RC mutants characterized by
a widely altered P+/P midpoint potential relative to wild-type [5].
In these mutants, the acceleration of P+QA
recombination induced
by cooling to 10 K in the dark decreased with increasing midpoint
potential [6]. Interestingly, incorporation into dehydrated trehalose
matrices causes instead acceleration of the kinetics by the same factor
for all P+/P midpoint potential values
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