I declare that the matter included in this thesis entitled "Studies on the Effect of Small Molecules on Protein Folding, Misfolding and Amyloidogenesis" is the result of research carried out by me at the Department of Biotechnology, Indian Institute of Technology, Guwahati, India under the supervision of Dr. . I am grateful to Dr. Rukhsani Chowdhury and Amartya from the Indian Institute of Chemical Biology, Calcutta, for their help with the MALDI TOF experiment.
Review of Literature .1 Protein Folding
Furthermore, studies have shown a strong correlation between destabilization of the conformation of the protein in the native state and its propensity to form amyloid (Chiti et al., 2000). Furthermore, pyrroloquinoline showed a significant inhibitory effect on the amyloid formation of mouse Aβ (1-42) and prion protein (Kim et al., 2009).
Scope of current research
Effect of sodium tetrathionate on egg white lysozyme amyloidogenesis was monitored by Thioflavin T assay and Atomic Force Microscopy imaging. Effect of sodium tetrathionate on amyloidogenesis of hen egg white lysozyme was monitored by Thioflavin T test and AFM.
Abstract
Introduction
Any factor that can increase the energy barrier between the folded state and the aggregated state will significantly slow down the aggregation process, giving the cellular quality control machinery enough time to direct the misfolded protein to the native conformation or to be degraded by proteosome complexes (Cohen and Kelly, 2003). This can be achieved in two ways either by stabilizing the misfolded state of the protein or by destabilizing the transition state that lies between the folded state and the aggregated state.
Materials & Methods .1 Materials
Upon reaching the plateau region (stationary phase of amyloid formation) it was found that the ThT intensity in the presence of the compounds approx. Of the three compounds, rottlerin was found to be the most effective inhibitor of both HEWL at pH 12.2 and Cyt C at pH 9.0 with an inhibitory efficiency of approx. Both sulconazole and clotrimazole significantly decreased the rate of unfolding of HEWL at pH 2.0 without changing the thermodynamic stability of the intermediate state (as shown from the equilibrium unfolding curve).
The upward curvature of the unfolding rate with GuHCl concentration suggests that the unfolding of HEWL at pH 2.0 in the presence of clotrimazole proceeds through parallel unfolding pathways (Fersht, 2000).
Conclusions
Abstract
Furthermore, amyloid formation has been found to be a generic property of all polypeptides, regardless of their source protein sequence or tertiary structure (Bucciantini et al., 2002). Previously, the effect of some compounds such as rottlerin, clotrimazole and sulconazole on the amyloidogenesis of selected prion proteins has been investigated (Feng et al., 2008). Furthermore, it is homologous to human lysozyme, variants of which lead to hereditary systemic amyloidosis (Pepsys et al., 1993).
Curcumin has been found to exhibit a disaggregation effect on preformed fibrils of HEWL at pH 2.0 after 2 days of incubation (Wang et al., 2009a).
Materials and Methods .1 Materials
Atomic Force Microscopy (AFM)
ANS binding assay
HEWL fibril formation after incubation in alkaline buffer was confirmed by thioflavin T fluorescence and rottlerin was added to the preformed fibril at 10-fold molar excess concentration. Changes in the HEWL fibril conformation in terms of exposed hydrophobic surface, upon addition of rottlerin, were investigated by ANS binding assay. This suggests that addition of rottlerin reduces the exposed hydrophobic surface in HEWL fibril and thus prevents ANS binding.
Further, changes in the secondary structure of the HEWL fibril upon the addition of rottlerin were monitored by FTIR spectra (Figure 3.5).
Discussions
The addition of rottlerin was followed by an immediate decrease in DNSCl anisotropy, indicating a decrease in the molecular size of the DNSCl-labeled HEWL fibril complex. Changes in surface hydrophobicity of HEWL fibrils upon addition of rottlerin were monitored by ANS fluorescence study. Furthermore, changes in the secondary structure of the HEWL fibril upon the addition of rottlerin were monitored by FTIR.
No additional peaks in the FTIR spectra of HEWL fibril-rottlerin complex and HEWL fibril alone suggest no covalent interaction between HEWL fibril and rottlerin.
Conclusions
To gain insight into the type of binding force associated with the interaction between HEWL and rottlerin at pH 12.2, techniques such as fluorescence quenching and FTIR were used. Quenching data for HEWL with rottlerin at different temperatures shows a decrease in quenching rate with increasing temperatures. This indicates the presence of hydrogen bonding or Van der Waals interaction between HEWL and rottlerin (Wang et al., 2009a).
This implies involvement of hydrogen bonding between HEWL and rottlerin interaction at pH 12.2 which is consistent with the quenching data.
Abstract
Introduction
Here we show that without sulfonation of the thiol group in HEWL, sodium tetrathionate causes complete inhibition of fibril formation at pH 2.0. Our results also indicate a critical role of the disulfide bond instead of the exposed hydrophobic surface in the protein in initiating fibrillogenesis in the early stages of fibril formation.
Materials and methods .1 Materials
The samples were then incubated in the dark for 30 min before a fluorescence emission scan was made from 400 nm to 600 nm at an excitation wavelength of 380 nm using the Cary Eclipse Fluorescence Spectrophotometer from Varian. The final concentration of the protein and denaturant in each sample was determined by spectrophotometry and refractive index measurements, respectively. Incubated samples were selectively excited at 295 nm for tryptophan and the emission was recorded between 315 nm and 400 nm using the Jobin-Yvon Fluoromax-3 spectrophotometer.
The unfolding kinetics was monitored by monitoring spectral changes in the intrinsic tryptophan fluorescence of the protein samples at 350 nm using the Cary Eclipse Fluorescence Spectrophotometer from Varian equipped with stopped flow equipment.
Results
The change in the tertiary structure of HEWL induced by the addition of STT and β-mercaptoethanol under amyloidogenic conditions was monitored through intrinsic fluorescence. Upon further incubation, no significant change was found in the intrinsic fluorescence of HEWL. Change in the exposed hydrophobic surface of HEWL upon addition of β-mercaptoethanol under amyloidogenic condition was also monitored through ANS fluorescence (Figure 4.11).
Initially, after 6 h of incubation, ANS fluorescence of HEWL in the presence of 1% (v/v) β-mercaptoethanol was significantly higher than the control sample, indicating the presence of a conformation with highly exposed hydrophobic surface.
Discussions
The effect of STT on the stability and unfolding kinetics of HEWL at pH 2.0 was studied. Thus, STT appeared to destabilize both the intermediate and transition states of HEWL at this pH to a similar extent as to keep the energy difference between the protein at pH 2.0 and the unfolded state unchanged. This suggests a role for the correct disulfide bond in stabilizing both the intermediate and transition states of the protein, which reside at pH 2.0 during the unfolding process.
Upon further incubation, there appears to be no significant change in λmax and fluorescence intensity of HEWL in the presence of STT and β-mercaptoethanol.
Conclusions
After 6 h of incubation, both STT and β-mercaptoethanol change the conformation of HEWL to a more solvent-exposed hydrophobic core conformation as seen by the red shift in λmax. Exposure of hydrophobic surface in HEWL by addition of 1% β-mercaptoethanol was investigated through ANS fluorescence (Figure 4.11). The study shows that β-mercaptoethanol induces structural change in HEWL, further exposing its hydrophobic surface.
However, it does not appear to promote full protein unfolding as evidenced by high ANS fluorescence.
Abstract
Introduction
The molecular mechanism of amyloid formation and the role of intermediate states during this transition remain elusive. Our results shed some light on the folding pathway of DKGR in specific and (α/β)8 barrel proteins in general. We identified an amyloidogenic molten globule-like state of DKGR and showed that molten globule-like intermediate states in proteins play a key role in amyloidogenesis.
Furthermore, we have shown that curcumin acts as a chaperone and inhibits amyloid formation by directly interacting with the amyloidogenic intermediate state and preventing aggregation.
Materials and methods .1 Materials
These fractions were then pooled and extensively dialyzed against 20 mM Tris buffer (pH 8.0) to remove salt present in the sample. These fractions were then pooled and extensively dialyzed against 50 mM phosphate buffer pH 7.0 to remove traces of NaCl. The protein stock solution was added to the appropriate buffer and the mixture was incubated for 24 hours at 25 °C.
Intrinsic fluorescence of the incubated samples was taken using the Jobin Yvon Fluoromax-3 Spectrofluorimeter with emission scanning off.
Results
For data collection, 10 µl of the sample was deposited on a freshly prepared thin layer of mica film and dried under nitrogen for 2 hours. The sample at pH 7.0 was added with 2 µl of 1 mM magnesium chloride to mica layer for effective adsorption.
Discussions
A range of human diseases are associated with protein amyloid formation leading to the malfunctioning of the cellular machinery (Thomas et al., 1995). The acrylamide quenching data also show two distinct curves for AI1 and AI2 indicating that these two are two distinct conformations, with the solvent exposure lying between that of the native folded state and fully denatured state. The molecular nature of the interactions of curcumin and the intermediate state is being studied in our laboratory.
A molten globule conformation has been found in prion oligomerization, and the conformational transition of the human prion protein from an α-helical to a β-sheet-rich structure is believed to be a critical event in prion pathogenesis (Gerber et al., 2007).
Conclusions
Summary*
These amyloids were found to be very similar in structural, immunological, toxic and tinctorial properties, although they originate from polypeptide chains that differ in sequence and tertiary structure. The inhibitory effect of these compounds on amyloidogenesis was found to be both polypeptide chain and pH specific, suggesting that amyloid inhibitor promiscuity appears to be elusive and that further studies in this direction are needed2. STT was found to facilitate intramolecular disulfide mixing in HEWL, resulting in the formation of HEWL species resistant to amyloid formation.
Curcumin was found to inhibit DKGR amyloidogenesis at a 1:1 molar ratio, suggesting a possible mode of action of curcumin through direct interaction with the polypeptide chain and prevention of aggregation.
Bibliography
Studies of the aggregation of mutant proteins in vitro provide insight into the genetics of amyloid diseases. Protofibrillar intermediates of amyloid beta protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. Inhibition of the formation of amyloid beta protein fibrils using biocompatible nanogels as artificial chaperones.
Curcumin inhibits the formation of amyloid beta oligomers and fibrils, binds plaques and reduces amyloid in vivo.
