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1.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
2.
J Colloid Interface Sci ; 602: 732-739, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34157514

RESUMO

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.


Assuntos
Lipídeos/química , Lipoproteínas HDL/química , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19 , Humanos , Glicoproteína da Espícula de Coronavírus/química
3.
Mol Syst Biol ; 17(5): e10280, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33943004

RESUMO

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

4.
Structure ; 29(9): 1003-1013.e4, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-33765407

RESUMO

Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.

5.
ACS Nano ; 15(4): 6709-6722, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33754708

RESUMO

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.


Assuntos
Nanopartículas , Apolipoproteínas E/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , Distribuição Tecidual
6.
Glycobiology ; 31(2): 151-158, 2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601663

RESUMO

l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host-pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L-1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.

7.
Nanomaterials (Basel) ; 10(12)2020 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-33291326

RESUMO

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.

8.
Cells ; 9(10)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977400

RESUMO

The experimental determination of the relative biological effectiveness of thermal neutron factors is fundamental in Boron Neutron Capture Therapy. The present values have been obtained while using mixed beams that consist of both neutrons and photons of various energies. A common weighting factor has been used for both thermal and fast neutron doses, although such an approach has been questioned. At the nuclear reactor of the Institut Laue-Langevin a pure low-energy neutron beam has been used to determine thermal neutron relative biological effectiveness factors. Different cancer cell lines, which correspond to glioblastoma, melanoma, and head and neck squamous cell carcinoma, and non-tumor cell lines (lung fibroblast and embryonic kidney), have been irradiated while using an experimental arrangement designed to minimize neutron-induced secondary gamma radiation. Additionally, the cells were irradiated with photons at a medical linear accelerator, providing reference data for comparison with that from neutron irradiation. The survival and proliferation were studied after irradiation, yielding the Relative Biological Effectiveness that corresponds to the damage of thermal neutrons for the different tissue types.


Assuntos
Terapia por Captura de Nêutron de Boro , Neoplasias/tratamento farmacológico , Nêutrons/uso terapêutico , Eficiência Biológica Relativa , Terapia por Captura de Nêutron de Boro/métodos , Raios gama , Humanos
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1865(10): 158769, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32712249

RESUMO

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as 'good' and 'bad' cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.


Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Lipídeos/genética , Lipoproteínas/genética , Aterosclerose/genética , Aterosclerose/patologia , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/genética , Gorduras na Dieta , Ácidos Graxos , Humanos , Lipoproteínas/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Triglicerídeos/genética , Triglicerídeos/metabolismo
10.
Appl Radiat Isot ; 163: 109205, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32392166

RESUMO

The cold neutron beam at the PF1b line at the Institut Laue-Langevin (ILL), without fast neutrons and a low contribution of gamma rays, is a very suitable facility to measure cell damage following low-energy neutron irradiation. The biological damage associated with the thermal and the boron doses can be obtained in order to evaluate the relative biological effectiveness (RBE) for Boron Neutron Capture Therapy. Three different experiments were carried out on the A375 melanoma cell line: the first one in a hospital LINAC, to obtain the reference radiation data, and the other two at the ILL, in which the damage to cells with and without boron compounds added was measured.

11.
Front Immunol ; 11: 601895, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33552059

RESUMO

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.


Assuntos
Multimerização Proteica , Fator H do Complemento/química , Fator H do Complemento/genética , Humanos , Domínios Proteicos
12.
Sci Rep ; 9(1): 8716, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213614

RESUMO

Following observations of survival of microbes and other life forms in deep subsurface environments it is necessary to understand their biological functioning under high pressure conditions. Key aspects of biochemical reactions and transport processes within cells are determined by the intracellular water dynamics. We studied water diffusion and rotational relaxation in live Shewanella oneidensis bacteria at pressures up to 500 MPa using quasi-elastic neutron scattering (QENS). The intracellular diffusion exhibits a significantly greater slowdown (by -10-30%) and an increase in rotational relaxation times (+10-40%) compared with water dynamics in the aqueous solutions used to resuspend the bacterial samples. Those results indicate both a pressure-induced viscosity increase and slowdown in ionic/macromolecular transport properties within the cells affecting the rates of metabolic and other biological processes. Our new data support emerging models for intracellular organisation with nanoscale water channels threading between macromolecular regions within a dynamically organized structure rather than a homogenous gel-like cytoplasm.


Assuntos
Citoplasma/metabolismo , Hidrodinâmica , Shewanella/metabolismo , Água/metabolismo , Transporte Biológico , Difusão , Cinética , Difração de Nêutrons/métodos , Nêutrons , Pressão , Shewanella/citologia , Viscosidade
13.
Sci Rep ; 9(1): 7591, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110185

RESUMO

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.


Assuntos
Lipídeos/fisiologia , Lipoproteínas/metabolismo , Membranas/metabolismo , Aterosclerose/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Nêutrons , Fosfatidilcolinas/metabolismo , Espalhamento a Baixo Ângulo
14.
Sci Rep ; 9(1): 5118, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914734

RESUMO

The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron scattering and neutron reflection, the level of deuteration of a sample can be varied to match the scattering length density of a specific D2O/H2O solvent mixture. This can be of major value in structural studies where specific regions of a complex system can be highlighted, and others rendered invisible. This is especially useful in analyses of the structure and dynamics of membrane components. In mammalian membranes, the presence of cholesterol is crucial in modulating the properties of lipids and in their interaction with proteins. Here, a protocol is described for the production of partially deuterated cholesterol which has a neutron scattering length density that matches that of 100% D2O solvent (hereby named matchout cholesterol). The level of deuteration was determined by mass spectrometry and nuclear magnetic resonance. The cholesterol match-point was verified experimentally using small angle neutron scattering. The matchout cholesterol was used to investigate the incorporation of cholesterol in various phosphatidylcholine supported lipid bilayers by neutron reflectometry. The study included both saturated and unsaturated lipids, as well as lipids with varying chain lengths. It was found that cholesterol is distributed asymmetrically within the bilayer, positioned closer to the headgroups of the lipids than to the middle of the tail core, regardless of the phosphatidylcholine species.


Assuntos
Colesterol/química , Óxido de Deutério/química , Bicamadas Lipídicas/química , Difração de Nêutrons , Espalhamento a Baixo Ângulo
15.
Nat Commun ; 10(1): 925, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804345

RESUMO

Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein.


Assuntos
Amiloidose/genética , Pré-Albumina/química , Pré-Albumina/genética , Amiloidose/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Pré-Albumina/metabolismo , Conformação Proteica , Dobramento de Proteína , Desdobramento de Proteína
16.
Commun Biol ; 1: 206, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30511020

RESUMO

Plasma-membrane Ca2+-ATPases expel Ca2+ from the cytoplasm and are key regulators of Ca2+ homeostasis in eukaryotes. They are autoinhibited under low Ca2+ concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca2+ pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme.

17.
Structure ; 26(8): 1072-1079.e4, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29937358

RESUMO

Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier" nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Espalhamento a Baixo Ângulo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Deutério/química , Deutério/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Difração de Nêutrons , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
18.
Langmuir ; 34(17): 5020-5029, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29629770

RESUMO

The application of protein deuteration and high flux neutron reflectometry has allowed a comparison of the adsorption properties of lysozyme at the air-water interface from dilute solutions in the absence and presence of high concentrations of two strong denaturants: urea and guanidine hydrochloride (GuHCl). The surface excess and adsorption layer thickness were resolved and complemented by images of the mesoscopic lateral morphology from Brewster angle microscopy. It was revealed that the thickness of the adsorption layer in the absence of added denaturants is less than the short axial length of the lysozyme molecule, which indicates deformation of the globules at the interface. Two-dimensional elongated aggregates in the surface layer merge over time to form an extensive network at the approach to steady state. Addition of denaturants in the bulk results in an acceleration of adsorption and an increase of the adsorption layer thickness. These results are attributed to incomplete collapse of the globules in the bulk from the effects of the denaturants as a result of interactions between remote amino acid residues. Both effects may be connected to an increase of the effective total volume of macromolecules due to the changes of their tertiary structure, that is, the formation of molten globules under the influence of urea and the partial unfolding of globules under the influence of GuHCl. In the former case, the increase of globule hydrophobicity leads to cooperative aggregation in the surface layer during adsorption. Unlike in the case of solutions without denaturants, the surface aggregates are short and wormlike, their size does not change with time, and they do not merge to form an extensive network at the approach to steady state. To the best of our knowledge, these are the first observations of cooperative aggregation in lysozyme adsorption layers.

19.
Colloids Surf B Biointerfaces ; 168: 126-133, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29433911

RESUMO

The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.


Assuntos
Deutério/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Difração de Nêutrons/métodos , Membrana Celular/química , Colesterol/química , Fosfolipídeos/química , Pichia/química
20.
Chem Phys Lipids ; 212: 80-87, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29357283

RESUMO

Deuteration of biomolecules has a major impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris using flask and fed-batch fermenter cultures in deuterated minimal medium. Perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated by neutron reflectometry measurements using the FIGARO instrument at the ILL.


Assuntos
Colesterol/análise , Difração de Nêutrons , Pichia/metabolismo , Proteínas Recombinantes/química , Reatores Biológicos , Colesterol/análogos & derivados , Deutério/química , Espectrometria de Massas , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo
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