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

More documents

Recommendations

Info

2364 Juárez et al. during the first 4 days of incubation as a result of the sequential unfolding of domains I and II of HSA. This period of time is slightly shorter than at pH 7.4 (mainly in the presence of electrolyte excess) because the expanded E state already involves an important alteration in the tertiary structure. In situ observation of fibrillar structures at acidic pH: TEM HSA samples obtained at different incubation times were also subjected to TEM analysis under acidic conditions. At room temperature in the absence of salt, no aggregates Biophysical Journal 96(6) 2353–2370 FIGURE 10 Time evolution of secondary structure compositions of HSA solutions at pH 3.0 at 25 C(a and b) or65C(c and d) in the absence and the presence of 50 mM NaCl, respectively. (:) a-helix, (-) b-turn, (C) unordered, and (;) b-sheet conformations. were observed during the first 2 days of the incubation process. From the third day, the presence of small clusters (globules) of aggregated protein could be observed. These aggregates have a globular or spherical shape, not the regular fibrillar appearance associated with amyloid structures (see Fig. 11 a). In addition, less numerous, more elongated aggregates (20–30 nm long, 3–4 nm wide) can be also observed, and their population slightly increases as incubation proceeds. The formation of these types of aggregates is characterized by a decrease of helical structure accompanied by a slight rise in b-sheet and unordered conformations from CD measurements at long incubation times. FIGURE 11 TEM pictures of the different stages of the HSA fibrillation process at pH 3.0 in the presence of 50 mM NaCl (a) at25C after 150 h of incubation, and at 65 C after (b) 24h,(c) 150 h, and (d) 250 h of incubation in the presence of 50 mM NaCl. 158
Fibrillation Pathway of HSA 2365 When the temperature is raised to 65 C, fibril formation takes place in several steps. First, formation of quasi-spherical aggregates takes place under incubation times similar to those at pH 7.4, and these seem to be fairly soluble. On the other hand, we have found that a coexistence of spherical aggregates with circular ring-shaped particles of larger size (~100–300 nm) is observed at short incubation times (on the order of a few hours) in the presence of excess electrolyte (Fig. 11 b). These structures appear as a possible intermediate structural rearrangement of smaller protein aggregates before fibril formation facilitated by electrostatic screening. There is no evidence as to whether structural reorganization to form fibrils takes place within this type of aggregates, or whether dissociation of HSA molecules from these structures occurs. Based on our CD data, which indicate that a progressive gaining of b-sheet structure and unordered conformation at the expense of a-helices takes place in the first part of the incubation process, we suggest that only after a certain critical amount of b-sheet structure is reached inside these ringshaped particles will short fibrils be formed in solution from decomposition of the former and become stable. Once sufficient molecules are present within the oligomer, reorganization steps become thermodynamically favorable as a result of an increase in the number of hydrogen bonds and other stabilizing interactions. Once fragments of highly ordered aggregates are present, the free energy for addition of monomeric molecules to a growing fibril will become more favorable. Similar circular ring-shaped structures have also been observed as a structural intermediate before the formation of amyloid fibrils in the self-assembly process of insulin (67), Ab17-42 peptide (68), Ab1-4 peptide, and HaPrP23- 144 prion protein (69). In this case, the circular structures may incorporate into fibrils but also self-aggregate to form large, amorphous structures (57). Given the large structural differences between HSA insulin and Ab17-42 peptide, and their distinct propensity to form amyloid fibrils, it seems reasonable to think that these circular ring-shaped structures can be a sort of common structural intermediate of amyloid fibril formation under different solution conditions. After longer incubation times (6 days) for HSA solutions in the absence of electrolyte at 65 C, small, well-defined, short protofibrils (~100 nm long and 3–4 nm wide) start to appear (Fig. 11 c). The corresponding CD spectrum shows a progressive increase in the proportion of b-structure at the expense of the helical one during incubation. In contrast, the presence of electrolyte involves an additional step: the formation of short curly fibers occurs in a broader incubation timescale than at physiological pH (Fig. 11 d); under further incubation, the fibrils appear to increase slightly in number and length. They range from ~100 nm to several hundred nanometers in length and 8 to 11 nm in width; thus, they are shorter on average than those obtained at physiological conditions but have the same average width. Closed fibril loops with diameters of ~100 nm were also frequently observed because the formed filaments can remain short and thin to enable them to bend and form closed rings, as also observed for b2 microglobulin (58), a-crystallin (56), equine lysozyme (70), insulin (67), and a-synuclein (71). This fibrillar material appears similar to protofilaments observed in the early stages of other amyloidogenic systems, given the small length and the absence of higher-order structures. DISCUSSION The ability to form amyloid fibrils is a generic property of polypeptide chains, although the propensity of different regions of proteins to form such structures varies substantially (72). The properties of unfolded polypeptides, including their relative propensities for a- and b-structure, their intrinsic solubility, and the nature of the interactions within the resultant fibrillar structures, are likely to be particularly important determinants in their relative abilities to form fibrils. The conformation of the partially folded state is not by itself a critical feature of fibril formation; rather, it is suggested that the basis for amyloidogenesis is the presence of partially denaturation conditions that destabilize the native fold of the protein but do not preclude noncovalent interactions between the various groups within the protein. In our case, it appears necessary to destabilize HSA molecules and hence to generate a partially folded intermediate that can aggregate to form fibrils. Thus, one can readily atttain conditions in which such aggregation-prone intermediate states are significantly populated by lowering the pH or raising the temperature. ThT fluorescence, CR staining, XRD, and TEM pictures demonstrate the formation of amyloid-like structures under these conditions. The aggregation process is governed by the balance between attractive and repulsive interactions between protein molecules. Conformational changes induced by heat increase the number of hydrophobic residues exposed to the aqueous solvent. The exposure of these groups results in attractive hydrophobic interactions that play a dominant role in the aggregation process. Repulsive forces are induced by a surface charge that can be modulated by changes in pH, which controls the net charge of the protein, and by the ionic strength of the solvent, which controls the screening of electrostatic interactions (73). Absence of a lag phase in HSA fibrillation process in most solution conditions A first characteristic of the HSA fibrillation process is the absence of a lag phase, as previously observed for bovine serum albumin (BSA) (74) and acyl phosphatase (75) under all solutions conditions analyzed except for acidic pH at room temperature. This occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. This is supported by the fact that seeding of preformed aggregates does not accelerate the fibrillation process. In this regard, it has been suggested that large multidomain proteins like BSA are able to form Biophysical Journal 96(6) 2353–2370 159
Page 1:
Grupo de Física de Coloides y Pol
Page 5:
Agradecimientos A mi Familia A mi P
Page 9:
v List of papers I. Self-assembly p
Page 12 and 13:
2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.
Page 14 and 15:
5.4 5.5 5.3.2 5.5.1 Chapter 6 Kinet
Page 16 and 17:
amiloides de la proteína albúmina
Page 18 and 19:
vesiculares son el resultado de la
Page 20 and 21:
origina debido a condiciones ambien
Page 22 and 23:
con las fibras vecinas más cercana
Page 25 and 26:
Abstract In the present work, we in
Page 27:
[Au]/[protein] molar ratio and numb
Page 30 and 31:
synthetic polymers are obtained fro
Page 32 and 33:
On the other hand, in terms of chem
Page 34 and 35:
Additionally, to obtain a better kn
Page 36 and 37:
therefore, characteristic of solid
Page 38 and 39:
presence of electrostatic charge in
Page 41 and 42:
Contents 2.1 2.2 2.3 2.4 2.5 2.6 2.
Page 43 and 44:
where w is the meniscus’ weight d
Page 45 and 46:
that they exert little force one on
Page 47 and 48:
This oscillating dipole acts as an
Page 49 and 50:
(APD) and its associated optics (pi
Page 51 and 52:
where r is the distance of the dipo
Page 53 and 54:
When , the former equation can be s
Page 55 and 56:
autocorrelation function (ACF). In
Page 57 and 58:
and, therefore, . The autocorrelati
Page 59 and 60:
A transmission electron microscope
Page 61 and 62:
2.5.- Atomic force microscopy (AFM)
Page 63 and 64:
ecause of the energy loss in the ex
Page 65 and 66:
Figure 2.15. Schematic representati
Page 67 and 68:
ackbone of proteins. Since proteins
Page 69 and 70:
According to classical mechanics, t
Page 71 and 72:
chemical composition, configuration
Page 73 and 74:
2.9.1.- Viscoelasticity Many materi
Page 75 and 76:
where is the total strain rate, whi
Page 77 and 78:
Using equations 2.40 and 2.41 in eq
Page 79 and 80:
scattering process that incoming X-
Page 81 and 82:
maximum intensity of the peak, Imax
Page 83 and 84:
translation of the reciprocal latti
Page 85 and 86:
For a single crystal, the chance to
Page 87 and 88:
excitation; namely, a valance elect
Page 89 and 90:
Figure 2.27. Distinction between si
Page 91 and 92:
microscope, there are two polarized
Page 93 and 94:
In particular, we use SQUID to meas
Page 95 and 96:
are aligned by means of an external
Page 97 and 98:
on the digital profile of a drop im
Page 99 and 100:
Laser confocal microscopy. Confocal
Page 101 and 102:
Fluorescence spectroscopy. Fluoresc
Page 103:
Superconducting quantum interferenc
Page 106 and 107:
temperatures, the chains are mixed
Page 108 and 109:
e detected by a given method. Therm
Page 110 and 111:
subsequently, of their thermodynami
Page 112 and 113:
a) O b) H 2C CH 2 O H 2C CH Figure
Page 115:
Contents 4.1 4.2 4.3 4.3.1 CHAPTER
Page 118 and 119:
7108 Langmuir, Vol. 24, No. 14, 200
Page 120 and 121:
7110 Langmuir, Vol. 24, No. 14, 200
Page 122 and 123: 7112 Langmuir, Vol. 24, No. 14, 200
Page 128 and 129: 15704 J. Phys. Chem. C, Vol. 114, N
Page 138 and 139: 4.3.1.- Relevant aspects I. For E12
Page 141 and 142: Contents 5.1 5.2 5.3 5.4 5.5 5.1.1
Page 143 and 144: acids whose lateral side chains cha
Page 145 and 146: adjacent segments of an antiparalle
Page 147 and 148: 5.2.1.- Unfolded state Over the las
Page 149 and 150: 5.2.4.- Energy landscape: protein a
Page 151 and 152: molecular medicine viewpoint, prote
Page 153 and 154: shows the typical nucleation-depend
Page 155 and 156: the energy landscape of the aggrega
Page 157: exposed loop region. The secondary
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: Fibrillation Pathway of HSA 2363 FI
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
Page 203 and 204: 5 10 15 20 25 30 35 40 45 50 below,
Page 205 and 206: 5 10 15 20 25 30 55 Figure 3: Selec
Page 207 and 208: 10 15 Soft Matter Cite this: DOI: 1
Page 209 and 210: 5 10 15 Soft Matter Cite this: DOI:
Page 211: 5 65. L. A. Sikkink, M. Ramirez-Alv
Page 214 and 215: ions would interact electrostatical
Page 216: pubs.acs.org/JPCL Figure 3. (a) Tim
Page 220 and 221: (54) Almeida, N. L.; Oliveira, C. L
Page 222 and 223:
netospirillum magneticum strain AMB
Page 224 and 225:
One-Dimensional Magnetic Nanowires
Page 226 and 227:
Page 228 and 229:
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
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?