Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p(2)H (pD) and show that gradually lowering the p(2)H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity - similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.
- MeSH
- fluidita membrány MeSH
- fotosyntéza * MeSH
- fotosystém I (proteinový komplex) metabolismus ultrastruktura MeSH
- fotosystém II (proteinový komplex) metabolismus ultrastruktura MeSH
- hrách setý metabolismus ultrastruktura MeSH
- koncentrace vodíkových iontů MeSH
- listy rostlin metabolismus MeSH
- maloúhlový rozptyl * MeSH
- neutronová difrakce * MeSH
- přenos energie MeSH
- tylakoidy metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.
Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the 'lubricant' for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome.
- MeSH
- archeální RNA chemie MeSH
- Haloarcula marismortui chemie MeSH
- malé podjednotky ribozomu archebakteriální chemie MeSH
- messenger RNA chemie MeSH
- neutronová difrakce MeSH
- simulace molekulární dynamiky * MeSH
- velké podjednotky ribozomu archebakteriální chemie MeSH
- Publikační typ
- časopisecké články MeSH
The nucleation and growth of crystalline ice during cooling, and further crystallization processes during re-warming are considered to be key processes determining the success of low temperature storage of biological objects, as used in medical, agricultural and nature conservation applications. To avoid these problems a method, termed vitrification, is being developed to inhibit ice formation by use of high concentration of cryoprotectants and ultra-rapid cooling, but this is only successful across a limited number of biological objects and in small volume applications. This study explores physical processes of ice crystal formation in a model cryoprotective solution used previously in trials on vitrification of complex biological systems, to improve our understanding of the process and identify limiting biophysical factors. Here we present results of neutron scattering experiments which show that even if ice crystal formation has been suppressed during quench cooling, the water molecules, mobilised during warming, can crystallise as detectable ice. The crystallisation happens right after melting of the glass phase formed during quench cooling, whilst the sample is still transiting deep cryogenic temperatures. We also observe strong water isotope effects on ice crystallisation processes in the cryoprotectant mixture. In the neutron scattering experiment with a fully protiated water component, we observe ready crystallisation occurring just after the glass melting transition. On the contrary with a fully deuteriated water component, the process of crystallisation is either completely or substantially supressed. This behaviour might be explained by nuclear quantum effects in water. The strong isotope effect, observed here, may play an important role in development of new cryopreservation strategies.
We report kinetic studies of therapeutically highly potent polymer-drug conjugates consisting of amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers bearing the anticancer drug doxorubicin (Dox). Highly hydrophobic cholesterol moieties as well as the drug were attached to the polymer backbone by a pH-sensitive hydrazone bond. Moreover, the structure of the spacer between the polymer carrier and the cholesterol moiety differed in order to influence the release rate of the hydrophobic moiety, and thus the disintegration of the high-molecular-weight micellar nanoparticle structure. We performed time-dependent SAXS/SANS measurements after changing pH from a typical blood value (pH 7.2) to that of tumor cells (pH 5.0) to characterize the drug release and changes in particle size and shape. Nanoparticles composed of the conjugates containing Dox were generally larger than the drug-free ones. For most conjugates, nanoparticle growth or decay was observed in the time range of several hours. It was established that the growth/decay rate and the steady-state size of nanoparticles depend on the spacer structure. From analytical fitting, we conclude that the most probable structure of the nanoparticles was a core-shell or a core with attached Gaussian chains. We concluded that the spacer structure determined the fate of a cholesterol derivative after the pH jump. Fitting results for 5α-cholestan-3-onecholestan-3-one and cholesteryl-4-oxopentanoate (Lev-chol) implied that cholesterol moieties continuously escape from the core of the nanoparticle core and concentrate in the hydrophilic shell. In contrast, cholest-4-en-3-one spacer prevent cholesterol escaping. Dox moiety release was only observed after a change in pH. Such findings justify the model proposed in our previous paper. Lastly, the cholesteryl 4-(2-oxopropyl)benzoate (Opb-Chol) was a different case where after the release of hydrophobic Opb-Chol moieties, the core becomes more compact. The physicochemical mechanisms responsible for the scenarios of the different spacers are discussed.
- MeSH
- akrylamidy chemie MeSH
- časové faktory MeSH
- cholesterol chemie MeSH
- difrakce rentgenového záření MeSH
- doxorubicin aplikace a dávkování MeSH
- hydrofobní a hydrofilní interakce MeSH
- hydrolýza MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny polymethakrylové chemie MeSH
- lékové transportní systémy * MeSH
- maloúhlový rozptyl MeSH
- micely * MeSH
- molekulární struktura MeSH
- neutronová difrakce MeSH
- povrchové vlastnosti MeSH
- protinádorové látky aplikace a dávkování MeSH
- velikost částic MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.
- MeSH
- akrylamidy chemie MeSH
- algoritmy MeSH
- anizotropie MeSH
- cholesterol MeSH
- difrakce rentgenového záření MeSH
- doxorubicin analogy a deriváty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- makromolekulární látky chemie MeSH
- maloúhlový rozptyl MeSH
- micely MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- nanokapsle chemie MeSH
- neutronová difrakce MeSH
- protinádorová antibiotika chemie MeSH
- světlo MeSH
- velikost částic MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Crystalline order of molded and then bi-axially stretched foils prepared from atactic PVC resin is investigated by means of wide-angle neutron diffraction (WAND). The observed high-resolution WAND patterns of all samples are dominated by a sharp maximum corresponding to the inter-planar distance 0.52 nm. Two weaker maxima are also resolved at 0.62 and 0.78 nm. Intensities of the peaks vary with deformation ratios of the samples and their diffraction position. Average size of the coherently scattering domains is estimated as approximately 4-8 nm. Based on the experimental data, a novel model of crystalline order of atactic PVC is proposed.
