Self-Assembly of Synthetic and Biological Polymeric Systems of ...

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properties induce solvent-adapted structural “responses” of the aggregating protein, which leads to a structural diversity of the resulting aggregates/fibers. 21-23 In particular, alcohols are included among the most commonly studied cosolvents. Several studies have shown that the effects of alcohol on very different systems and processes such as the thermal denaturation of nucleic acids and proteins, protein folding, micellization of surfactant molecules, or the solubility of nonelectrolytes are strikingly similar. 24-26 Also, water/alcohol mixtures at moderately low pH values have been suggested as model systems for studying the joint action of the local decrease in both pH and dielectric constant on the protein structure near the membrane surface; 27-32 similarly, new ways of protein administration and delivery based on inhalation systems with non-aqueous solvents as suspension media also reinforce the necessity of clarifying how the presence of the solvent affects the protein conformation and biological 24, 25 activity. From a thermodynamic standpoint, the protein accessible surface area (ASA), the solvent exposure of hydrophobic residues, and a solvophobic backbone 33 can be taken as barriers hampering protein-solvent interactions. 34 Therefore, the hierarchical assembly of amyloids can perfectly be understood as an alternative to the native packing conformational struggle of a polypeptide chain, reducing ASA and saturating hydrogen bonding, while a simultaneous decrease in the configurational entropy of the protein is offset by gains in solvent entropy. The role of hydrational forces in protein aggreation in vivo, while still poorly understood, may be instrumental in elucidating the molecular basis of amyloidosis and, as such, attracts considerable interest. 35 In this way, the propensity for amyloid formation in mixed solvents of different proteins as, for example, human serum albumin (HSA), hen egg white lysozyme, bovine βlactoglobulin, histone H2A, insulin, and bovine trypsinogen amongst many others, has been characterized. 36-39 Amongst them, HSA has been proposed as a good model for protein aggregation studies 40-42 as a consequence of both its physiological implications as a carrier protein and blood pressure regulator, together with its propensity to easily aggregate in vitro. Previous reports have addressed the HSA fibrillation kinetics, fibrillation pathway, and intermediate and final protein aggregated structures under varying pH and ionic strength solution conditions. 40-42 However, a detailed characterization of the aggregation/fibril process and the structure of the resulting aggregates in mixed solvent solutions under varying external conditions is still lacked. In the present work, we present a detailed study on the aggregation/fibrillation pathway of the protein human serum albumin (HSA) in water/ethanol mixed solvent solutions at varying pH and temperature conditions. This enables us to make a clear comparison between the fibrillation pathway and intermediate/final protein aggregates in the mixed solvent with those obtained in pure aqueous solution in order to define the role of hydrational forces on the protein fibrillation mechanism. In fact, changes on the fibrillation mechanism from a downhill to a nucleated-growth polymerization mechanism at acidic pH and high temperature solution conditions are confirmed. Overall, as a result of differences in packing depending on the solution condition, different structures for the resulting fibrils are observed, from classically thin and very long straight to shorter 5 10 15 20 25 30 35 40 45 50 55 and more curly fibers. In this regard, it comes as no surprise that factors favoring or disfavoring exposure of solvophobic groups must affect thermodynamic preferences for particular conformations. 2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] 186 60 65 Materials and Methods Materials. Human serum albumin (70024-90-7), and Thioflavin T (ThT) were obtained from Sigma Chemical Co. ThT was used as 70 received. All other chemicals were of the highest purity available. 75 80 85 90 95 100 105 110 115 Preparation of HSA solutions. HSA was purified by liquid chromatography using a Superdex 75 column equilibrated with 0.01 M phosphate buffer before use. To prepare the protein solutions, increasing volumes of ethanol were added to 1.0 mL of HSA stock solution either at pH 7.4 (sodium monophosphate-sodium diphosphate buffer) or pH 2.0 (glycine + HCl buffer), to get the desired ethanol concentration in the mixed solvent. The final protein concentration in all cases was 10 mg/mL with ionic strength 10 mM. pH was kept constant by adding HCl or NaOH when needed. The protein concentration was determined spectrophotometrically, using a molar absorption coefficient of 35219 M -1 cm -1 at 280 nm. 43 Aliquots were added through an electronic disperser unit Dosimat Metrohm 765. Experiments were carried out using double distilled, deionized and degassed water. Before incubation, solutions were filtered through a 0.2 µm filter into sterile test tubes. Samples were incubated at a specified temperature in a refluxed reactor for 15-20 days as required. Light Scattering. DLS and SLS intensities were measured at 25 °C by means of an ALV-5000F (ALV-GmbH) instrument working with a vertically polarized incident light of wavelength λ = 488 nm supplied by a CW diode-pumped Nd; YAG solid-state laser supplied by Coherent, Inc. and operated at 400 mW. The intensity scale was calibrated against scattering from toluene. Measurements were carried out at a scattering angle θ = 90° to the incident beam. Solutions were equilibrated for 30 min before measurements to reach thermal stabilization. Experiment duration was in the range of 3-5 min, and each experiment was repeated two or more times. The correlation functions from DLS were analyzed using the CONTIN method to obtain intensity distributions of decay rates (Γ). 44 The decay rate distribution functions gave distributions of apparent diffusion coefficient (D app = Γ /q 2 ,where the scattering vector q = (4πns/λ)sin(θ /2), and ns is the refractive index of solvent) and integrating over the intensity distribution gave the intensity-weighted average of D . app Values of the apparent hydrodynamic radius (r , radius of h,app hydrodynamically equivalent hard sphere corresponding to D ) app were calculated from the Stokes-Einstein equation:
5 10 15 20 25 r h,app = kT/(6πηD app ) (1) where k is the Boltzmann constant and η is the viscosity of water at temperature T. Thioflavin T spectroscopy. Protein and ThT were dissolved in buffer at a final proteindye molar ratio of 50:1. Samples were continuously stirred only during data acquisition intervals in order to avoid ThT deposition onto protein aggregates/fibrils. Fluorescence was measured in a Cary Eclipse fluorescence spectrophotometer equipped with a temperature controller and a multi-cell sample holder (Varian Instruments Inc.). Excitation and emission were at wavelengths of 450 and 482 nm, respectively. All intensities were backgroundcorrected for the ThT-fluorescence in the respective solvent in the absence and presence of monomeric HSA at pH 7.4 and 2.0. Circular Dichroism (CD). Far-UV circular dichroism (CD) spectra were obtained using a JASCO-715 automatic recording spectropolarimeter with a JASCO PTC-343 Peltier-type thermostated cell holder. Quartz cuvettes with 0.2 cm pathlength were used. The protein concentration was reduced to 0.1 mg/mL in order to avoid light scattering effects as much as possible due to protein aggregation. CD spectra were obtained from aliquots withdrawn from the aggregation mixtures at the indicated conditions after incubation, and recorded at wavelengths between 195 and 300 nm at 25 ºC. The mean residue ellipticity θ (deg cm 2 dmol -1 30 ) was calculated 35 40 45 50 55 according to: θ=(θObs/10)/(MRM/lc), where θobs is the observed ellipticity (in deg), MRM is the mean residue molecular mass (in g mol -1 ), l is the optical path-length (in cm), and c is the protein concentration (in g ml -1 ). The secondary structure of the protein was calculated from far-UV CD spectra by running the SELCON3, CONTIN, and DSST programs within the DICHROWEB server. 45,46 Final results were assumed when data generated from all programs showed convergence. Transmission electron microscopy (TEM). HSA solutions were applied to carbon-coated copper grids, blotted, washed, negatively stained with 2% (w/v) of phosphotungstic acid, air dried, and then examined with a Phillips CM-12 transmission electron microscope operating at an accelerating voltage of 120 kV. Samples were diluted between 20-200-fold prior deposition onto the grids. Results and Discussion To induce HSA aggregation and subsequent amyloid formation we use 10 mg/mL HSA solutions at either pH 7.4 or pH 2.0 and ionic strength of 10 mM. At pH 7.4 the protein is in its native state whereas at pH 2.0 it is in its E-expanded one, which is characterized by a separation of domain III and a loss of the intradomain helices of domain I. 47 Monomer sizes and structural compositions were similar to those previously reported. 40 Samples were prepared by adding specified volumes of protein stock solutions and different amounts of ethanol, which has been shown to destabilize both the secondary and tertiary structures of HSA; 36 subsequently, samples were incubated at room or elevated temperature (65 ºC, above the melting temperature of HSA) 48,49 to induce further protein structural changes and aggregation. This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 3 187 60 65 70 75 80 85 90 95 100 105 110 Aggregate formation. The formation of HSA aggregates and their aggregation kinetics were firstly analyzed by SLS. Figure 1 shows the time evolution of the scattered light intensity of HSA samples at varying pH, temperature and alcohol content conditions upon incubation. Different curve profiles are obtained depending on incubation conditions. At physiological pH (Figure 1a-b), the plots reflect a quick increase in the scattered intensity at short incubation times, which denotes the fast formation of protein aggregates. SLS plot profiles suggest a mechanism of continuous protein aggregation (in particular, a continuous fibrillation process as later confirmed by ThT, CD and TEM data) without a nucleation step, as denoted the absence of a lag phase in the early periods of incubation (see inset in Figure 1a). In addition, the absence of a faster aggregation process when protein seeds (i.e. protein aggregates) are added to protein solutions followed by subsequent incubation, and the decrease in the aggregation rate if the starting protein concentration is lowered (see Figure S1 in ESI) further indicate that under the present conditions HSA aggregation occurs by means of a classical coagulation or downhill polymerization mechanism, i.e. each protein monomer association is bimolecular, effectively irreversible and thermodynamically favorable, so that there is no energy barrier to impede aggregate growth. 50 A similar behavior has been previously obtained in the absence of alcohol. 40-42,50,51 ThT fluorescence data confirmed similar trends as those observed by SLS (see Figure S2). High values of ThT fluorescence point to an important formation/gain of β-sheet structure along the protein aggregation process, which is a first evidence of the possible formation of HSA fibrils during the aggregation process, 37 as further confirmed below by CD and TEM techniques. Also, under physiological conditions we observe that as the ethanol concentration increases up to 60% (v/v) the formation of more numerous and/or bulkier protein aggregates takes place as derived from both the increments in scattered intensities – proportional to protein concentration and molecular weight (Figure 1a,b), and the shifts of the aggregate size distributions to larger values (see Figure 2a-b below). The clustering of alcohol molecules and the enhancement of alcohol-water interactions at intermediate ethanol concentrations in the mixed solvent should modify the extent of protein hydration and molecular packing 53 as confirmed by CD and FT-IR below, favoring an enhanced protein aggregation. At larger alcohol concentrations (> 60% (v/v)), protein solutions became progressively turbid as a consequence of the formation of even larger aggregates, 52 which gradually precipitated (see below for further explanation).
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Grupo de Física de Coloides y Pol
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Agradecimientos A mi Familia A mi P
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v List of papers I. Self-assembly p
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2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.
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5.4 5.5 5.3.2 5.5.1 Chapter 6 Kinet
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amiloides de la proteína albúmina
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vesiculares son el resultado de la
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origina debido a condiciones ambien
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con las fibras vecinas más cercana
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Abstract In the present work, we in
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[Au]/[protein] molar ratio and numb
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synthetic polymers are obtained fro
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On the other hand, in terms of chem
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Additionally, to obtain a better kn
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therefore, characteristic of solid
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presence of electrostatic charge in
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Contents 2.1 2.2 2.3 2.4 2.5 2.6 2.
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where w is the meniscus’ weight d
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that they exert little force one on
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This oscillating dipole acts as an
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(APD) and its associated optics (pi
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where r is the distance of the dipo
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When , the former equation can be s
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autocorrelation function (ACF). In
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and, therefore, . The autocorrelati
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A transmission electron microscope
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2.5.- Atomic force microscopy (AFM)
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ecause of the energy loss in the ex
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Figure 2.15. Schematic representati
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ackbone of proteins. Since proteins
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According to classical mechanics, t
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chemical composition, configuration
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2.9.1.- Viscoelasticity Many materi
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where is the total strain rate, whi
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Using equations 2.40 and 2.41 in eq
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scattering process that incoming X-
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maximum intensity of the peak, Imax
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translation of the reciprocal latti
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For a single crystal, the chance to
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excitation; namely, a valance elect
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Figure 2.27. Distinction between si
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microscope, there are two polarized
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In particular, we use SQUID to meas
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are aligned by means of an external
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on the digital profile of a drop im
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Laser confocal microscopy. Confocal
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Fluorescence spectroscopy. Fluoresc
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Superconducting quantum interferenc
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temperatures, the chains are mixed
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e detected by a given method. Therm
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subsequently, of their thermodynami
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a) O b) H 2C CH 2 O H 2C CH Figure
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Contents 4.1 4.2 4.3 4.3.1 CHAPTER
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7108 Langmuir, Vol. 24, No. 14, 200
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7110 Langmuir, Vol. 24, No. 14, 200
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7112 Langmuir, Vol. 24, No. 14, 200
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7114 Langmuir, Vol. 24, No. 14, 200
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7116 Langmuir, Vol. 24, No. 14, 200
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15704 J. Phys. Chem. C, Vol. 114, N
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4.3.1.- Relevant aspects I. For E12
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Contents 5.1 5.2 5.3 5.4 5.5 5.1.1
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acids whose lateral side chains cha
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adjacent segments of an antiparalle
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5.2.1.- Unfolded state Over the las
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Page 179 and 180: Influence of Electrostatic Interact
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Page 187: Fibrillation Process of Human Serum
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Page 233 and 234: Conclusions The work has two parts:
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Page 237 and 238: References 1. Hiemenz, Paul C. Poly
Page 239 and 240: 33. Wang, Z. L. HANDBOOK OF MICROSC
Page 241 and 242: 67. Polymers Get Organized. Bucknal
Page 243 and 244: 94. Are there Pathways for Protein
Page 245 and 246: 123. Designing conditions for in vi
Page 247: 151. SERS-based diagnosis and biode
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