I am also grateful to all my seniors (Dr. Mahanty, Dr. Sahu, Dr. Majumder, Dr. Gopi, Dr. Purama, Dr. Shiva, Dr. Atul, Dr. Ballav, Dr. Sandeep, Dr. Dilip, en Dr. Ramakrishna), al my groepmaats (Dr. Pallav, Dr. Sanjay, Dr. Achlesh, Dr. Shampa, Dr. Vigya) en all koshuisvriende (Narsihn, Gaurav, Kaushik, Beda, Sukhi, Shadab, Vijay Mishra, Himanshu jee , Arvind, Sandip, Zella, Rajendra, Yata, Asim, K. Hegdge, Sourabh, Ratual, Naresh, Kasturi, Rachna en Sachi) en al my M.Tech vriende. This showed that henliosiem amorfe en geordende (fibrillére) aggregate in pH 12.2 by kamertemperatura vorm. Daar is vir die eerste keer gegen dat HEWL beide bolformiede aggregate en fibrille in pH 12.2 by kamertemperatura vorm.
Experimental materials, techniques and conditions 34
Decrease in size of hen egg white lysozyme aggregates with decrease in monomer concentration from micro to
Protein aggregation
- History of amyloid deposits
- The Structure of amyloid fibrils
- Structural studies using high resolution techniques
- The polymorphism of amyloid fibrils
- Mechanism of protein aggregation
- Nucleated growth dependent amyloid formation
- Structured Oligomers and protofibrils
- Unstructured oligomers and protofibrils
- Protein aggregation through isodesmic polymerization
- Protein conformation versus protein aggregation .1 Globular protein aggregation from partial unfolding
- Aggregation through native like oligomers
- Changes in sequence influences amyloid formation
Proteins require the correct folded conformation to perform their specific biological functions (Anfinsen et al., 1973). Later, the repeating nature of the cross-linked beta-sheet structure of amyloids was tested by X-ray fiber diffraction (Sunde et al., 1997). At equilibrium, the final amount of monomer would exist in equilibrium with the polymer (Harper et al., 1997).
Inhibition of protein aggregation
Macromolecule interaction exhibiting high compensatory charge can affect the aggregation of polypeptide chains (Fernandez et al., 2004, Giasson et al., 2003). Saccharide molecules such as trehalose at very high concentration were used to suppress polyglutamine diseases (Tanaka et al., 2004). The further approach is to target proteolytic cleavage such as γ-secretase, β-secretase (Citron et al., 2002, John et al., 2003) for Aβ aggregation.
Lysozyme aggregation
- Aggregation of hen lysozyme in vitro
- Using acidic pH
- Using ethanol
- Using guanidine hydrochloride
- Using other conditions
- Inhibition of lysozyme aggregation
- Motivation and objectives of this work
It is necessary and useful to obtain detailed information in this regard (Swaminathan et al., 2011). The inhibitory effect of p-benzoquinone and the endogenous neurohormone melatonin has also been observed against HEWL amyloid formation at acidic pH ( Wang et al., 2006 ). Arginine was recently found to control heat-induced aggregation at the isoelectric point of HEWL ( Tomita et al., 2011 ).
Methods .1 Stock solutions
These additives 14 mM SDS, 3 mM CTAB and 20 mM DTT were prepared in sodium dihydrogen phosphate buffer, pH 12.2 from their respective stock solution. For characterization of HEWL aggregates after preliminary incubation with chitotriose and NAG, the 50 mM chitotriose and 250 mM NAG stock solution was prepared in deionized water (MilliQ). Later, it was diluted into 100 µM HEWL containing >1.5 mM chitotriose (from an overnight incubated sample) in pH 12.2 buffer and left for incubation to observe the effect on aggregation. D) Sample in the presence of NAG: Stock solution of 250 mM NAG was prepared in deionized water (MilliQ) immediately before initiation of overnight incubation in 10 mM phosphate buffer at pH 7.3.
Dissolved HEWL was stirred using rice magetic beads and 100 µL dabcyl succinimidyl ester added slowly. For the complete reaction sample continuously was stirred
Time resolved fluorescence intensity decay and anisotropy decay
As previously mentioned, the fluorescence lifetime is the average amount of time the fluorophore spends in their excited state before emission. After the arrival of the first photon from the sample, the multichannel analyzer (MCA) converts the voltage from the TAC to the time channel using an analog to digital converter (ADC). The raw (G-factor corrected) anisotropy decays were tail fitted using a sum of two exponentials (see equation two) extracting the slower rotational correlation time (φ2) corresponding to global motion of the HEWL aggregate.
Tryptophan fluorescence quenching
Before the time-resolved anisotropy measurements of the HEWL aggregate sample were taken, G-factor was determined using ANS in ethanol, where 0.13 ns rotational correlation time (φ1) was found. Since the 0.15 ns IRF pulse width is negligible compared to the time scale of protein rotational motion (> 4 ns), the extracted values of φ2 by this tail-fit approach are not affected by the consequences of IRF folding. = 0.5 mM), reducing the fluorescence intensity possibly due to their paramagnetic nature, which promotes fluorophore to undergo intersystem crossing to the triplet state, whereas heavy atom such as iodide exhibits their quenching behavior by spin-orbit coupling. Because of its modification and larger size, iodide is used to study surface accessibility of fluorophore in macromolecules.
Collisional quenching occurs when an excited fluorophore comes into contact with an atom or molecule that can facilitate a nonradiative transition to the ground state. In the above equation, F0 and F are the fluorescence intensities observed in the absence and presence of the quencher, respectively, kq is the bimolecular quenching constant, τ0 is the unquenched lifetime of the fluorophore, and [Q] is the quencher concentration. In the simplest case, a plot of F0/F vs. [Q] should give a straight line with a slope equal to KSV.
The fluorescence of tryptophan was quenched by adding KI (0 to 0.8 M) as described earlier (Kumar et al., 2007). To prevent the formation of iodate (I-3), 0.1 mM sodium thiosulfate was added in stock solution of KI. A fixed concentration (1.2 µM) of aggregated lysozyme was diluted into cuvettes for 120 µM or 3 µM monomer concentrations, while for 0.3 µM HEWL, KI was added directly.
ANS binding
Thioflavin T binding
Atomic force microscopy
It also reduces the lateral (side to side) force on the sample because the tip spends less time on the surface of the sample. MAC Mode (Magnetic Alternating Current Mode): Cantilever coated with magnetic material is required for this imaging mode. Prior to sample preparation, freshly cleaved mica was cleaned using cellotape as much as possible by peeling method.
In the next step, HEWL samples (10 µl) were applied to freshly cleaved mica in the presence of 10 mM Mg 2+ , washed with 0.2 µm filtered deionized water after several minutes to remove unadsorbed sample, and dried under nitrogen. HEWL incubated in the presence of SDS and CTAB was eluted through Amersham PD-10 columns to remove free surfactants before sample preparation for imaging. The sample in the presence of chitotriose and NAG was diluted 10-fold in incubation buffer before sample preparation.
Samples were imaged in air under AAC or MAC MODE (non-contact) on PICO PLUSTM AFM purchased from Molecular Imaging, USA. Cantilever type PPP-MFMR-20 (resonance frequency, 60-70 kHz; . Nanosensors) was used for MAC mode, while type PPP-NCL-50 (resonance frequency, 150 kHz; Molecular Imaging) was used for AAC mode. Images were acquired digitally at a scan rate of 1–2 lines/second with 256 data points per line.
Size exclusion chromatography .1 Matrix preparation and column packing
Free thiol estimation and blocking
- Size of aggregates
- Morphology of aggregates
Monitoring the aggregation of chicken egg white lysozyme at pH 12.2 at room temperature in the absence and presence of additives such as surfactants (SDS, CTAB) and DTT. A previous study showed that HEWL aggregates slowly (Homchaudhuri et al., 2006) at pH 12.2 at room temperature. Similarly, the effect of surfactants and DTT on the size/shape of HEWL aggregates at pH 12.2 was not investigated.
Based on previous finding, size/shape of HEWL at pH 12.2 in absence and in presence of SDS, CTAB and DTT was investigated using gel filtration experiment. After size heterogeneity analysis, it will be worthwhile to observe the morphology of HEWL aggregates in pH 12.2 in absence and presence of surfactant (SDS, CTAB) and DTT. Interestingly, this is the first report of spontaneous formation of fibrillar structure of HEWL in pH 12.2 at room temperature.
In the presence of 14 mM SDS (Fig. 3.2.2 F), phase images of HEWL-SDS complex presented as an outer layer of bound micellar SDS to HEWL aggregates. Some groups have found fibrils at pH 9.2 in the presence of surfactant (Moosavi-Movahedi et al., 2007). The AFM images can track the conglomeration of HEWL monomers in the presence of surfactants.
Overall, an elution peak at ∼20 mL indicates that the protein is reasonably smaller and compact in the presence of CTAB compared to SDS.
Conclusions
- Size of aggregates
- Helical structure
- Accessibility of sulfhydryl groups
- Morphology
To obtain comparative information regarding the size and heterogeneity of HEWL oligomers in the presence of additives, size exclusion chromatography experiments were performed. It is worthwhile to observe the helical conformation of HEWL in the presence of additives such as chitotriose. Previous study has shown that HEWL activity changes in the presence of NAG, chitotriose and control samples (Kumar et al., 2008).
Therefore, based on this hypothesis, we proceeded to measure the accessibility of free thiol groups of HEWL in the presence of NAG and chitotriose. Thus, it appears that reduced but significant growth of aggregates also occurs in the presence of NAG or chitotriose. Similarly, HEWL, in the presence of NAG at pH 12.2, was shown in Fig.
These results are supported by the high fluorescence intensity observed with ThT in HEWL pH 12.2 and in the presence of NAG (Kumar et al., 2008). However, neither the ordered fibrillar HEWL nor large amorphous aggregates were exhibited in the presence of chitotriose, although small oligomers (∼150 nm × 50 nm) were found. It will be worthwhile to discuss the results where change in size of aggregates, exposure of free [–SH], helical content and morphology were observed in the presence of chitotriose, NAG and control.
In the presence of NAG, gel filtration data show a heterogeneous size of aggregates, which can be either larger or smaller aggregates, even at a later time of incubation (208 h).
Conclusions
- Onset of aggregation measured by fluorescence resonance energy transfer (FRET)
- Changes in local environment inside aggregates
- HEWL [µM],
- Accessibility of sulfhydryl group among aggregates
- Morphology of aggregates is concentration independent
- Model for HEWL aggregation at pH 12.2
- Conclusion
- Alteration of exposed hydrophobic regions
- Presence of oligomers/fibrils
- Size and dynamics of aggregates
- Morphology of aggregates
- Conclusions
The average accessibility of six trp residues during growth of HEWL aggregates at pH 12.2 was tested by quenching of trp fluorescence using KI (Eftink, et al., 1991). The fitted anisotropy growth parameters at pH 12.2 for different HEWL concentrations are shown in Table 5.2.3.1. The overall results clearly suggest that at pH 12.2: a) the rotational dynamics of the dansyl probe slows down with time due to aggregate growth and b) growth-dependent slowing.
Hen lysozyme contains four disulfide bonds which are prone to cleavage after transfer to pH 12.2 and may remain as eight free –SH groups. Assays of free thiols in HEWL (see Fig. 5.2.4 A, B and C) reveal that the formation of aggregates during 96 h incubation at pH 12.2 is not stabilized by disulfide bonds, unlike at later time points (Kumar et al ., 2008). . Exposure to pH 12.2 causes rapid partial unfolding of the native protein M, resulting in monomer B with exposed hydrophobic regions.
Structure and dynamics of s-carboxyamidomethylcysteine derivative aggregates of hen egg white lysozyme in alkaline pH 12.2 at room temperature. If –SH blocks, HEWL is likely to form globular aggregates at pH 12.2, and then further investigation of size and rotational dynamics of whole aggregates is required. For this experiment, similar dansyl-HEWL conjugates and thiol-blocked HEWL conjugates were used and the sample was incubated in pH 12.2 at 301 K.
These data suggest that when free. of aggregates in pH 12.2 decreases compared to unblocked HEWL. The AFM images were acquired after incubation of thiol-blocked and free thiol HEWL in pH 12.2 for different durations. Interestingly, ThT and AFM results are consistent and suggest that fibril formation was suppressed after alkylation of free thiol by HEWL in pH 12.2 at room temperature.
