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5 10 15 20 25 Figure 4: Normalized maximum ThT fluorescence intensities of HSA samples incubated at different ethanol concentrations in the mixed solvent at a) pH 7.4 and 65 ºC, b) pH 7.4 and 25 ºC, c) pH 2.0 and 65 ºC and d) pH 2.0 and 25 ºC. Normalization is made with respect to the maximum ThT fluorescence observed at pH 7.4 and 65 ºC. 30 ethanol concentrations larger than 50% (v/v) the β-sheet content increases, as denoted from the progressive shift of the minimum in CD spectra to 215 nm, which is characteristic of β-strand formation. In contrast, at acidic pH and 25 ºC (Figure 3d), the structural content of protein samples at ethanol concentrations 35 lower than 60% (v/v) remains almost invariable. Since HSA molecules are in a starting acid-denaturated state, alcohol stabilizes the α-helix conformation of the acid-unfolded protein monomers by minimizing the exposure of the peptide backbone. In particular, at alcohol concentrations < 40% (v/v), 40 intermolecular interactions are disfavored by suppression of the strong aggregate-stabilizing effect of negatively charged residues, still resulting in an effective electrostatic repulsion between positively charged chains. Hence, under these conditions alcohol stabilizes the molten-globule state of HSA during incubation, and only protein clusters can be formed. 63,64 45 The presence of these clusters, confirmed by the presence of a small peak at relatively low aggregate sizes (∼20-30 nm) (see Figures 2d), implies some increase in light scattering from solution as shown previously and, thus, possibly originates the slight decrease in ellipticity 50 observed in Figure 3d. Protein molecules experience additional structural rearrangements at ethanol concentrations between 50- 90% (v/v) when the polarity of the medium is drastically changed. This involves a decrease in α-helix structure and formation of β-strands that, as observed from light scattering 55 data, are prone to aggregate. FT-IR measurements corroborate the structural changes undergone by protein molecules depicted by CD data (for additional comments see ESI). Although reliable in detecting β-strands and probing hydrogen bonding between them, CD and FT-IR spectroscopies fail to 60 discriminate between amorphous and ordered aggregates. Since ThT dye strongly emits when bound to amyloid-like material rather than to amorphous aggregates, we conducted ThT fluorescence measurements of HSA samples under the different solution conditions in order to determine the presence of ordered 65 aggregates (amyloid-like fibrils) in solution. Experimental data were background corrected for solvent and monomeric protein contributions and normalized to the largest value attained, i.e., at pH 7.4 and 65 ºC at 70 % (v/v) ethanol. Figure 4 confirms that βsheet rich structures (fibrils as shown by TEM pictures below) are 70 formed under all conditions except at acidic pH and 25 ºC at alcohol concentrations below 60% (v/v). At pH 7.4 and 65 ºC (Figure 4a), the amount of fibrils progressively increases up to an ethanol content of 70% (v/v), and then decreases. This decrease in ThT fluorescence can be associated with the presence of 75 mature fibril association/aggregation, as confirmed by TEM (see below), which may reduce the protein exposed surface area and the number of available ThT binding sites. 65 In contrast, at acidic conditions (Figure 4c) the maximum level of fibril formation takes place at the largest ethanol concentrations (70-90% v/v). On 80 the other hand, the temporal evolution of ThT binding confirms 8 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] 192
10 15 Soft Matter Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx a) c) d) e) f) Dynamic Article Links ► PAPER Figure 5: Selected TEM images of HSA samples incubated at pH 7.4 and a-c) 65 ºC, or d-f) 25 ºC at ethanol concentrations in the mixed solvent of a,d) 20, b,e) 60, c,f) 80% (v/v), respectively. the trends previously observed from SLS kinetic data, although some differences at acidic and high temperature conditions are observed (see Figure S2 and further explanation in ESI). Based on all the above findings, the combination of i) incubation at high temperature and ii) the presence of ethanol as a cosolvent in solution result in important variations of the protein structural content and subsequent strengthening of hydrophobic interactions between protein molecules. Incubation at high temperatures involves the denaturation of protein monomers due to the disruption of the structure of protein domains 48,49,63 while the presence of the cosolvent changes the solvent polarity and This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 9 193 5 20 25 30 b) induces a further exposure of residues, 66 which enhances the internal degrees of freedom of side-chains. 67 Then, in most of the studied conditions, the cosolvent induces additional modifications in the starting protein structure, leading to a strong protein aggregation and fibril formation, but with differences in the packing mechanisms leading to fibril polymorphism, as will be shown below. All these facts become even clearer when comparing the secondary structure compositions here obtained with those measured under identical incubation conditions in the absence of alcohol. 36,40-42
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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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5.2.4.- Energy landscape: protein a
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molecular medicine viewpoint, prote
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shows the typical nucleation-depend
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Page 161 and 162: Biophysical Journal Volume 96 March
Page 163 and 164: Fibrillation Pathway of HSA 2355 q
Page 165 and 166: Fibrillation Pathway of HSA 2357 mo
Page 167 and 168: Fibrillation Pathway of HSA 2359 in
Page 169 and 170: Fibrillation Pathway of HSA 2361 Fu
Page 171 and 172: Fibrillation Pathway of HSA 2363 FI
Page 173 and 174: Fibrillation Pathway of HSA 2365 Wh
Page 175 and 176: Fibrillation Pathway of HSA 2367 of
Page 177 and 178: Fibrillation Pathway of HSA 2369 17
Page 179 and 180: Influence of Electrostatic Interact
Page 181 and 182: Fibrillation Process of Human Serum
Page 187: Fibrillation Process of Human Serum
Page 190 and 191: 12392 J. Phys. Chem. B, Vol. 113, N
Page 199 and 200: 5 10 15 20 25 30 35 40 45 Soft Matt
Page 201 and 202: 5 10 15 20 25 r h,app = kT/(6πηD
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Page 211: 5 65. L. A. Sikkink, M. Ramirez-Alv
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Page 216: pubs.acs.org/JPCL Figure 3. (a) Tim
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Page 222 and 223: netospirillum magneticum strain AMB
Page 224 and 225: One-Dimensional Magnetic Nanowires
Page 230 and 231: temperatures, and solvent polarity.
Page 233 and 234: Conclusions The work has two parts:
Page 235 and 236: equire a highly organized and unsta
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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