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1.
Sci Rep ; 13(1): 16516, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37783694

RESUMO

Bacterial colonization is mediated by fimbriae, which are thin hair-like appendages dispersed from the bacterial surface. The aggregative adherence fimbriae from enteroaggregative E. coli are secreted through the outer membrane and consist of polymerized minor and major pilin subunits. Currently, the understanding of the structural morphology and the role of the minor pilin subunit in the polymerized fimbriae are limited. In this study we use small-angle X-ray scattering to reveal the structural morphology of purified fimbriae in solution. We show that the aggregative fimbriae are compact arrangements of subunit proteins Agg5A + Agg3B which are assembled pairwise on a flexible string rather than extended in relatively straight filaments. Absence of the minor subunit leads to less compact fimbriae, but did not affect the length. The study provides novel insights into the structural morphology and assembly of the aggregative adherence fimbriae. Our study suggests that the minor subunit is not located at the tip of the fimbriae as previously speculated but has a higher importance for the assembled fimbriae by affecting the global structure.


Assuntos
Escherichia coli , Proteínas de Fímbrias , Proteínas de Fímbrias/metabolismo , Escherichia coli/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Aderência Bacteriana , Fímbrias Bacterianas/metabolismo
2.
Front Mol Biosci ; 7: 125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754613

RESUMO

Interactions between proteins and surfactants are both of fundamental interest and relevant for applications in food, cosmetics and detergency. The anionic surfactant sodium dodecyl sulfate (SDS) denatures essentially all proteins. Denaturation typically involves a number of distinct steps where growing numbers of SDS molecules bind to the protein, as seen in multidisciplinary approaches combining several complementary techniques. We adopt this approach to study the SDS-induced unfolding of Ca2+-depleted α-lactalbumin (aLA), a protein particularly sensitive toward denaturation by surfactants. By combining stopped-flow mixing of protein and surfactant solutions with stopped-flow synchrotron small-angle X-ray scattering (SAXS), circular dichroism (CD) and Trp fluorescence, together with information from previous calorimetric studies, we construct a detailed picture of the unfolding process at the level of both protein and surfactant. A protein-surfactant complex is formed within the dead time of mixing (2.5 ms). Initially a cluster of SDS molecules binds asymmetrically, i.e., to one side of the protein, after which aLA redistributes around the SDS cluster. This occurs in two kinetic steps where the complex grows in number of both SDS and protein molecules, concomitant with protein unfolding. During these steps, the core-shell complex undergoes changes in shell thickness as well as core shape and radius. The entire process is very sensitive to SDS concentration and completes within 10 s at an SDS:aLA ratio of 9, decreasing to 0.2 s at 60 SDS:aLA. The number of aLA molecules per SDS complex drops from 1.9 to 1.0 over this range of ratios. While both CD and Trp kinetics reveal a fast and a slow conformational transition, only the slow transition is observed by SAXS, indicating that the protein-SDS complex (which is monitored by SAXS) adjusts to the presence of the unfolded protein. We attribute the rapid unfolding of aLA to its predominantly α-helical structure, which persists in SDS (albeit as isolated helices), enabling aLA to unfold without undergoing major secondary structural changes unlike ß-sheet rich proteins. Nevertheless, the overall unfolding steps are broadly similar to those of the more ß-rich protein ß-lactoglobulin, suggesting that this unfolding model is representative of the general process of SDS-unfolding of proteins.

3.
J Appl Crystallogr ; 51(Pt 5): 1455-1466, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279642

RESUMO

In small-angle scattering theory and data modeling, it is generally assumed that each scattered ray - photon or neutron - is only scattered once on its path through the sample. This assumption greatly simplifies the interpretation of the data and is valid in many cases. However, it breaks down under conditions of high scattering power, increasing with sample concentration, scattering contrast, sample path length and ray wavelength. For samples with a significant scattering power, disregarding multiple scattering effects can lead to erroneous conclusions on the structure of the investigated sample. In this paper, the impact of multiple scattering effects on different types of scattering pattern are determined, and methods for assessing and addressing them are discussed, including the general implementation of multiple scattering effects in structural model fits. The modification of scattering patterns by multiple scattering is determined for the sphere scattering function and the Gaussian function, as well as for different Sabine-type functions, including the Debye-Andersen-Brumberger (DAB) model and the Lorentzian scattering function. The calculations are performed using the semi-analytical convolution method developed by Schelten & Schmatz [J. Appl. Cryst. (1980 ▸). 13, 385-390], facilitated by analytical expressions for intermediate functions, and checked with Monte Carlo simulations. The results show how a difference in the shape of the scattering function plotted versus momentum transfer q results in different multiple scattering effects at low q, where information on the particle mass and radius of gyration is contained.

4.
FEBS J ; 285(2): 357-371, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29178440

RESUMO

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.


Assuntos
Detergentes/química , Glucosídeos/química , Maltose/análogos & derivados , Proteínas de Membrana/química , Difração de Nêutrons , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Espalhamento a Baixo Ângulo , Maltose/química , Micelas , Conformação Proteica
5.
J Pept Sci ; 23(12): 845-854, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29057588

RESUMO

Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight-lowering effects of bariatric surgery involve stimulated secretion of appetite-regulating gut hormones, including glucagon-like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux-en-Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux-en-Y gastric bypass. We therefore developed novel secretin-based peptide co-agonists and identified a lead compound, GUB06-046, that exhibited potent agonism of both the secretin receptor and glucagon-like peptide 1 receptor. Semi-acute administration of GUB06-046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06-046 to diabetic db/db mice for 8 weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta-cell mass after GUB06-046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06-046 on appetite regulation, glucose homeostasis, and beta-cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Índice Glicêmico/efeitos dos fármacos , Obesidade/tratamento farmacológico , Peptídeos/administração & dosagem , Secretina/química , Animais , Cirurgia Bariátrica , Proliferação de Células , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Obesidade/metabolismo , Obesidade/cirurgia , Peptídeos/farmacologia , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores dos Hormônios Gastrointestinais/agonistas , Secretina/metabolismo
6.
Membranes (Basel) ; 5(3): 307-51, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26264033

RESUMO

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

7.
Nano Lett ; 15(2): 1158-65, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25555128

RESUMO

The synthesis, characterization, and electrokinetic energy conversion performance have been investigated experimentally in a charged polymeric membrane based on a blend of nitrocellulose and sulfonated polystyrene. The membrane is characterized by a moderate ion exchange capacity and a relatively porous structure with average pore diameter of 11 nm. With electrokinetic energy conversion, pressure can be converted directly into electric energy and vice versa. From the electrokinetic transport properties, a remarkably large intrinsic maximum efficiency of 46% is found. It is anticipated that the results are an experimental verification of theoretical models that predict high electrokinetic energy conversion efficiency in pores with high permselectivity and hydrodynamic slip flow. Furthermore, the result is a promising step for obtaining efficient low-cost electrokinetic generators and pumps for small or microscale applications.


Assuntos
Colódio/química , Membranas Artificiais , Nanoporos , Poliestirenos/química , Sulfonas/química , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
Angew Chem Int Ed Engl ; 53(43): 11524-8, 2014 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-25197008

RESUMO

Despite over a century of modern surfactant science, the kinetic pathways of morphological transitions in micellar systems are still not well understood. This is mainly as a result of the lack of sufficiently fast methods that can capture the structural changes of such transitions. Herein, a simple surfactant system consisting of sodium dodecyl sulfate (SDS) in aqueous NaCl solutions is investigated. Combining synchrotron radiation small-angle X-ray scattering (SAXS) with fast stopped-flow mixing schemes allows monitoring the process where polymer-like micelles are formed from globular micelles when the salt concentration is suddenly increased. The results show that "worm-like" micelles are formed by fusion of globular micelles and short cylinders in a fashion that bears similarities to a step-like polymerization process.

9.
J Biol Chem ; 288(32): 23518-28, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23775077

RESUMO

Intermediate amyloidogenic states along the amyloid ß peptide (Aß) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aß aggregation, we here investigate surfactant-induced Aß aggregation. This process leads to co-aggregates featuring a ß-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aß in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via ß-structure to the fully formed α-helical state at high surfactant concentration. The ß-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aß co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with ß-structure promoting substances, such as surfactants, Aß is kinetically driven toward an aggregation-prone state.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Modelos Químicos , Fragmentos de Peptídeos/química , Tensoativos/química , Animais , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
10.
Biochim Biophys Acta ; 1828(8): 1909-17, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23567913

RESUMO

We have in this study investigated the composition, structure and spectroscopical properties of multilamellar vesicles composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and up to 10mol% of triolein (TO), a triglyceride. We found in agreement with previous results that the mixtures with 10mol% TO spontaneously separate into two distinct phases, heavy (HF) and light (LF), with different densities and found this also to be the case for 2 and 5mol% TO. The compositions of the two phases were investigated by quantitative lipid mass spectrometric analysis, and with this method we found that TO had a solubility maximum of about 4mol% in the HF, whereas it was markedly up-concentrated in the LF. Electron paramagnetic resonance spectroscopy indicated POPC membranes of all tested concentrations of TO in both phases to be almost unperturbed by the presence of TO and to exist as vesicular structures containing entrapped water. Bilayer structure of the membranes was supported by small angle X-ray scattering that showed the membranes to form a lamellar phase. Fluorescence spectroscopy with the polarity sensitive dye Nile red revealed, that the LF samples with more than 5mol% TO contained pure TO domains. These observations are consistent with an earlier MD simulation study by us and our co-workers suggesting triglycerides to be located in lens shaped, blister-like domains between the two lipid bilayer leaflets (Khandelia et al. (2010) [26]).


Assuntos
Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Triglicerídeos/química , Trioleína/química , Espectroscopia de Ressonância de Spin Eletrônica , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Fosfatidilcolinas/metabolismo , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/metabolismo , Trioleína/metabolismo
11.
J Am Chem Soc ; 135(19): 7214-22, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23590205

RESUMO

Self-assembly of amphiphilic molecules into micelles occurs on very short times scales of typically some milliseconds, and the structural evolution is therefore very challenging to observe experimentally. While rate constants of surfactant micelle kinetics have been accessed by spectroscopic techniques for decades, so far no experiments providing detailed information on the structural evolution of surfactant micelles during their formation process have been reported. In this work we show that by applying synchrotron small-angle X-ray scattering (SAXS) in combination with the stopped-flow mixing technique, the entire micelle formation process from single surfactants to equilibrium micelles can be followed in situ. Using a sugar-based surfactant system of dodecyl maltoside (DDM) in dimethylformamide (DMF), micelle formation can be induced simply by adding water, and this can be followed in situ by SAXS. Mixing of water and DMF is an exothermic process where the micelle formation process occurs under nonisothermal conditions with a temperature gradient relaxing from about 40 to 20 °C. A kinetic nucleation and growth mechanism model describing micelle formation by insertion/expulsion of single molecules under nonisothermal conditions was developed and shown to describe the data very well.

12.
J Colloid Interface Sci ; 397: 9-17, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23484773

RESUMO

Alkaline sodium silicate solutions with SiO2:Na2O molar ratios in the range 4-10 are known to be colloidally unstable manifested in phase separation or gelation. The mechanistic understanding of this instability is generally poor. To improve this situation the microscopic structure of a series of solutions with ratios in the range 3.3-8.9 has been characterised using small-angle X-ray scattering, Dynamic light scattering, Fourier transformed infrared spectroscopy, and (29)Si Nuclear magnetic resonance spectroscopy to cover the relevant length scales related to silica clusters, aggregates, and particles present. In the starting solution, with ratio 3.3, there are silica present in three fractions. The main part is present as small silica clusters with a radius of 0.7 nm. There are also a significant portion of monomers/small oligomeric silica species as well as a minute amount of larger colloidal silica particles. At a higher SiO2:Na2O ratio, above approximately 4, smaller spherical colloidal particles are formed due to condensation reactions. However, as a result of a too high ionic strength the suspension is not stable and the particles aggregate to fractal structures with a size that depends on ratio and ageing time. At the highest SiO2:Na2O ratio, fractals are not formed because of the lower ionic strength and the smaller colloidal particles are stable in the solution. By carefully adding small amounts of NaCl to the high ratio solution it is possible to induce gelation of the solution confirming the hypothesis that the instability region is due to too high electrolyte concentration for the silica species present under those conditions.

13.
Biomacromolecules ; 13(11): 3668-77, 2012 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-23082770

RESUMO

We report a facile method of generating ultradense poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) surface by using high temperature alone, which in turn provides dramatic improvement in resisting nonspecific bioadsorption. X-ray photoelectron spectroscopy (XPS) revealed that the surface graft density increased ~4 times higher on the surface prepared at 80 °C compared to 20 °C. The studies from small-angle X-ray scattering (SAXS) and the effect of varying ionic strength during/post assemblies at 20 and 80 °C indicated that the "cloud point grafting effect" is not the cause for obtaining high density grafting. Stringent long-term bioresistance tests have been conducted and the temperature-induced PLL-g-PEG surfaces have achieved (1) zero mammalian cell adsorption/migration for up to 36 days and (2) extremely close-to-zero protein adsorptions have been observed even after 36 days in 10% serum media and 24 h in whole blood within the ultrasensitive detection limit of time-of-flight secondary ion mass spectrometry (ToF-SIMS).


Assuntos
Materiais Revestidos Biocompatíveis/química , Polietilenoglicóis/química , Polilisina/análogos & derivados , Transplantes , Adsorção , Animais , Sangue , Células Cultivadas , Humanos , Concentração Osmolar , Espectroscopia Fotoeletrônica , Polilisina/sangue , Polilisina/química , Propriedades de Superfície , Temperatura
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