The Amyloid Fibrils Formation - Lab Report Example

The Amyloid Fibrils Formation and Amorphous Aggregation in Concanavalin A in Vitro Results and Discussion Results: At 250C: In pH 5.0 and 7.0, the fluorescence of tyrosine remained unchanged while at pH 9.0 intensity decreased significantly after 40h indicating conformational change and/or quenching of tyrosine fluorescence. Overall fluorescence of tyrosine at this pH remained lower than at other two pH values tested (Fig 1.a) Fig 1. Fluorescence intensities of a) tyrosine b)Tryptophan at and c) Thioflavin T at 25 0c and three different pH values. The fluorescence intensities for these three were measured at 303, 348, 482 nm respectively, in all the experiments. a) b) c) There was slight increase in Tryptophan fluorescence at pH 5.0 and 7.0. But at pH 9.0 the intensity decreased sharply from beginning up to 70h and then attained a plateau. The decrease of 400u indicated huge conformational change leading to buried and /or quenched Trp residues. Th T fluorescence decreased slightly up to 50 hrs and increased sharply thereafter reaching at peak at 80 hrs and then decreasing considerably. At pH 7 the intensity increased from beginning, reaching a max at 50h and decreasing sharply, thereafter. The Th T fluorescence shows reversible trend in these experiments and conformational changes are occurring fast. At pH 9.0, there was considerable increase in fluorescence intensity after 75h showing fibrillation (fig 1 c). At 37.2 0C: While at pH 5.0 and 7.0, the Intensities did not vary much but at pH 9.0 it decreased considerably after 50 h. showing decreased fibrillation or quenching of tyrosine (Fig 2 a). Fig 2. Fluorescence intensities of a) Tyrosine, b) Tryptophan and c) ThioflavinT at 37.2 0C Fig 2. a) Fig 2 b). Fig 2 c) Fig 2 b. shows increase in Trp fluorescence after 70 h at Ph 5.0, while at other pH values, there was no increase from the original values. At pH 9.0 clear and considerable increase in Thioflavin T intensity was observed, which is a positive indication for amyloid formation (Fig 2c). At 40 0C: Highest intensity was observed at pH 5.0 while considerably high Intensity at pH 7.0. However at pH 9.0 there was negligible intensity for tyrosine. The latter temperature and pH combination either create conditions for Tyrosine quenching or the aggregates deeply bury this amino acid as a result of conformational changes (Fig 3a). Fig 3. Fluorescence intensities of a) Tyrosine b) Tryptophan and c) Thioflavin T in Con A , at 40 0C a) b) c) Fig 3 b also shows considerable Trp fluorescence at pH 5.0 and 7.0 while at pH 9.0, intensity remained insignificant again showing buried/ quenched Trp residues. After a lag of 40 h the ThT intensity increased sharply to high level at pH 5.0. At pH 7.0, the plateau was attained after slight increase up to 20 h. At pH 9.0 the intensity increased at 70 h. Amyloid A40 Assay: Fig 4. Fluorescence of a) Tyrosine and b) Thioflavin T in A40 at 250 C Fig 4 a) The ThioflavinT bound to A increases at 40 h decreases thereafter and increases indicating conformational changes. Fig 4 b) Fig 4. shows Tyr intensity decreasing after 60h and so is the Th T intensity however the latter increases thereafter showing increased amyloidal fibril formation but at this time the Tyr seems to be quenched somewhat. Fig 5. TE Micrographs of Con A in pH 5.0 at 0 h Fig 5 a) X10K some amorphous aggregates are already present at this pH Fig 6. TEM showing Con A in pH 5.0 at a) 4 (X40K) and, b) 48h (X40K) c) 48 h at pH 7.0 (X15K and d) 48h at pH 9.0 at 25 0C Fig 6 a) only amorphous aggregates are present Fig 6 b) Fibril formation is clearly visible. Fig 6 c) 48h at pH 7.0 (X15K) Fig 6 d) 48 h at pH 9.0 (X10K) as expected the long amyloid fibrils are in state of formation Fig 7. Con A at 37.2 0C and pH 5.0 after 24h The physiological temperature and low pH shows abundant short rods Fig 8. A40 at 25 0 C a) 0, and b) 3h. (X20K) neither shows fibrils despite positive ThT fluorescence. Fig 7 shows AB40 as small nuclei at 24 h which are seen to coalesce at 72 hrs. Fig 7 A40 at a) 24, and b) 48h (X10K) Fig 7 a) Fig 7 b) Fig 8. A40 at a) 72, and b) 98 h (X10/3K) Fig 8 a) Fig 8 b) Only large amorphous structures seen after 72 h while a solitary long fibril is seen in the upper region after 96h (Fig 8 b). It is contrary to expectation for A40, which forms amyloidal aggregates and experimental procedure needs to be reviewed. Discussion: Under destabilizing condition of pH, temp, salt conc. conformational changes may lead to different aggregation pathways, thus resulting in structurally different supramolecular aggregates, such as amorphous aggregates or amyloid fibrils. In particular, the nature of the aggregation process can be significantly affected by pH, since the latter can determine the initial structure and the charge of the amino acids in the protein and, therefore, the degree of different kinds of interactions. In particular, Con A protein is basically a tetramer at physiological pH and a dimer at pH values less than 6. At pH between 8 and 9, Con A undergoes an apparent irreversible conformational transition, resulting in protein aggregation and precipitation (Booth et al 1997; Arai and Horai 1999; Hamada and Dobson, 2002). Dobson (2004) explained protein folding or misfolding structures of native proteins as the most stable structures that the main chain can assume. These are mainly defined by the side chain interactions. If the solution pH, temperature etc. in which the proteins are present is, however, such that these side chain interactions are not stable, the structures can unfold and sometimes, under many circumstances, reassemble in the form of amyloid fibrils. Thus, conditions favouring formation of amyloid fibrils requires a precondition whereby proteins involved are at least partially unfolded (Kelly, 1998). The proteins contain aromatic amino acids viz. tryptophan, tyrosine and phenylalanine which show fluorescence. When both Trp and Tyr are present the fluorescence is mainly due to Trp (Guilbault, 1990). In Fig 1 and 2 in this work is also seen similar trend as much higher intensity for Trp compared to Tyr. Though, intensity of Trp is high between pH values 9 to 10 but at 40 0C we observed negligible intensity at pH 9.0 (Fig 3). This clearly shows that Con A has undergone conformational change resulting in buried or quenched Trp residues. At high pH quenching of Trp may occur due to OH- ions. The conformational change to fibril formation is also supported by increased Th T fluorescence at this pH. At pH 9.0, though, Trp and Tyr fluorescence was not significant yet the ThT fluorescence showed clear increase reaching maxima after 90 h. The TEM photograph at 250 C and after 40 h also shows long amyloid fibrils at pH 9.0 (Fig). This observation is in agreement with that of Vetri et al (2007) who found that Th T fluorescence intensity increased only for the samples at the pH 8.9. with a predominance of amorphous aggregation at low pH, close to the isoelectric point of the protein, and amyloidal aggregation at high pH . Fig 6 indicated that fibril formation is supported at high pH but at low pH (5.0) when temperature is in physiological range the amyloid like rods are formed by Con A (Fig 7). In this experiment the Th T fluorescence increased at all pH values tested at 25 0C. Though TEM pictures showed only amorphous aggregates at pH 5.0 and 7.0. The increase in ThT fluorescence despite formation of amorphous aggregates could be explained by a different mechanism of its binding to Con A as suggested by Khurana et al. (2005). They posit that Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to detect amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay (10-20 M). They suggested that the micelles of ThT formed in aqueous solvent might be hydrogen-bonding to amyloid fibrils leading to changes in excitation spectrum. Later they demonstrated clearly that the micelles of thioflavin T bind the amyloid fibrils possibly involving both ionic and hydrophobic interactions. Since Th T binds nucleic acid also so it is the charged interaction and no specificity to beta structures. At low pH (5.0) the amorphous aggregates formed as can be seen by TEM at 0 and 4h while keeping sample longer at this pH does induced fibril formation as in TEM taken at 48h. The physiological temperature caused short rod formation at pH 5.0 as the TEM picture depicts abundant short fibrils. Our results are in agreement with Vetri et al who showed at pH 5.0, there were generally amorphous aggregates and sometimes very short rod like structures just as we too observed. The occurrence of fibrillar aggregates at pH8.9, thus confirming that the increase in Th T fluorescence observed at this pH is due to the formation of amyloid fibrils. Fibril height, obtained from the height in cross section measured from AFM images, is 2.40.2nm. This value is comparable to those measured for amyloid fibrils of other proteins imaged in air by AFM (Relini et al , 2004). But our result differ from Vetri et al (2007) who observed that the light scattering measurements value obtained at pH5.1 is much larger than the expected protein size; this indicates that aggregates are already present at this pH; on the contrary, within the experimental errors, their value obtained at pH8.9 is compatible with the absence of aggregation but we observed amorphous aggregates at pH 9.0. The increase in Th T fluorescence when the TEM picture shows amorphous aggregates can be explained by Kudou et al (2004) who assumed that heat induced aggregate of Con A could be porous structure allowing the binding to ANS and these heat-induced aggregates of Con A contain large amount of beta-sheet [Maeda et al 1989], implying that the aggregates of Con A may be stabilized by b-sheet interactions as well as hydrophobic interactions. Most likely, the increase in fluorescence of ThT is attributed to the enlargement of b-sheet content on the surface of aggregates. This is highly related to the increase in size of pore on this surface. The binding of ThT to these beta sheet structures on the porous surface of amorphous aggregate could have occurred in this experiment as well. The other explanation for increase in ThT fluorescence in conditions of amorphous aggregate formation is already given above. Read More