Using ANS fluorescence, we observed the gradual increase in exposure of the hydrophobic regions of lysozyme at pH 12.2, but not in the presence of DTT. Circular dichroism of lysozyme at pH 12.2 under different experimental conditions shows reduced helix content compared to native lysozyme except in the presence of SDS.
Concluding remarks
Introduction and literature review
Proteins
A characteristic three-dimensional structure and overall dynamics of protein is essentially required for their specific activity and stability (Daniel et al., 2003). Fluorescence anisotropy of dansyl chloride-conjugated egg white lysozyme has been used to monitor nanosecond dynamics of protein aggregation at alkaline condition ( Homchaudhuri et al., 2006 ).
Non-native protein
- Residual structures
- Protein aggregation
Residual structures in reduced lysozyme, which contains six hydrophobic clusters, significantly influence the formation of amyloid fibrils (Mishima et al., 2007). 3-(N-(4-bromophenyl)sulfamoyl)benzamide (C2-8) has been found to inhibit polyglutamine aggregation in Huntington's disease and suppress neurodegeneration in vivo (Zhang et al., 2005).
Work done by me
We applied sensitive fluorescence techniques to detect the growth HEWL in pH 12.2 alone and in the presence of detergents (SDS, CTAB) and DTT. We also monitored HEWL secondary structure in a month-old sample incubated at pH 12.2 alone and in the presence of additives at room temperature using circular dichroism (CD).
Absorption
Here Bab is the transition rate per unit energy density of the radiation while I(ν) is the energy density in the sample at frequency ν. The absorbance is linearly related to the concentration of the solution which can be expressed as.
Fluorescence
- Factors affecting fluorescence intensity
- Fluorescence kinetics
- Steady-state fluorescence
- Instrumentation for time-resolved fluorescence measurement
- Analysis of time-resolved decay data Time-resolved intensity decay analysis
As mentioned earlier, the fluorescence lifetime is the average amount of time a fluorophore spends in an excited state before emission. The instrument response function (IRF) [L(tk)] is the response of the instrument to a sample with zero fluorescence lifetime.
Electrophoresis
Native gel electrophoresis - In this mode, protein separation occurs in the absence of any denaturing agent. SDS-SIDE – Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis is a commonly used technique for separating protein in the presence of anionic detergent.
SDS PAGE– Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis is commonly used technique to separate protein in presence of anionic detergent
Since protein-SDS complexes have uniform charge to mass ratio, protein with higher molecular weight retains higher negative charge. This higher negative charge provides higher mobility of protein in SDS-PAGE gel which experiences increased frictional resistance. Because of their small size, bromophenol blue is totally unretarded and thus indicates the electrophoresis front.
Material and methods
- Chemical used
- Solutions prepared (A) Stock solution of proteins
- Denaturation of proteins
- Fluorescence quenching experiment
- Steady-state anisotropy measurement
- Chemical used
- Reagents prepared (A) Buffers used
- Procedure for labeling lysozyme with dansyl probe
- The aggregation reaction
- Effect of SDS, CTAB and DTT on HEWL aggregation
- Steady-state fluorescence anisotropy measurements
- Time-resolved fluorescence lifetime and anisotropy measurements
- Scattering experiment
- ANS binding assay
- Thioflavin T binding assay
- Estimation of free thiol groups
- Circular Dichroism spectroscopy
- HEWL assay
- Tricine-Sodium dodecyl sulphate polyacrylamide gel electrophoresis 16% Tricine-SDS-PAGE was performed as described by Schagger
- Peptide bond absorption
- Chemical used
- Reagents prepared (A) Buffers used
- Binding of chitotriose or NAG with HEWL
- The aggregation reaction
- ANS binding assay
- ThT binding assay
- Sodium dodecyl sulphate polyacrylamide gel electrophoresis Stacking gel (5%)
- HEWL assay
- Chemical used
- Reagents prepared (A) Buffer prepared
- Absorption of ThT with different surfactants
- Emission of ThT with different surfactants
- Determination of Critical Micellar Concentration (CMC) of surfactants Pyrene fluorescence emission and its peak III to peak I vibronic band intensity
- Quantum yield calculation
- Mammalian cell culture
- Fluorescence microscopy
- Lysozyme fibril formation
- Atomic force microscopic imaging
The absorption of fresh HEWL in deionized water and pH 12.2 buffer itself was also recorded with the same dilution. 50 mM, pH 7 phosphate buffer; 10 mM, pH 7.3 phosphate buffer; A 50 mM, pH 12.2 phosphate buffer and a 20 mM, pH 8.5 Gly-Gly buffer were prepared by dissolving the respective salts in deionized water and the appropriate pH was adjusted using a pH meter with the addition of 10 N NaOH or 10 N HCl. 50 mM, pH 7 phosphate buffer; A 50 mM, pH 12.2 phosphate buffer and a 20 mM, pH 8.5 Gly-Gly buffer were prepared by dissolving the respective salts in deionized water and the appropriate pH was adjusted using a pH meter with the addition of 10 N NaOH or 10 N HCl.
Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins
The unquenched fluorescence lifetime of trp in above proteins (except BSA and tetrapeptide) at 6 M GdnCl was obtained from the previous work of Swaminathan et al., 1994. The lifetime of tryptophan in BSA and tetrapeptide at 6 M GdnCl after overnight incubation was consistent with a previous measurement performed elsewhere. Later, the same experiment using 295 nm source was performed in our laboratory and we obtained almost the same average fluorescence lifetime of BSA and tetrapeptide as we obtained from TIFR, Mumbai.
108,111,123 Trp-Met-
Like many other trps in hydrophobic clusters, W59 in RNase T1 is also buried in a hydrophobic core of the native protein. W53 in barstar is also known to be buried in the hydrophobic core (Swaminathan et al., 1996). The absence of correlation between indole emission maxima and the rate of iodide quenching is evident in the case of the tetrapeptide, which has the lowest emission maximum and highest bimolecular quenching constant.
Effect of SDS, CTAB and DTT on the size, dynamics, activity and growth of soluble lysozyme aggregates
Light scattering experiment
After monitoring the growth of dansyl-conjugated HEWL at pH 12.2, we tried to observe the same phenomenon with unlabeled lysozyme in the same experimental condition. Increase in scattering intensity up to 360 minutes of pH 12.2 incubated HEWL indicates the formation of aggregates, while at pH 7, fairly constant values reflect the presence of monomeric lysozyme. This finding from diffusion experiments supports our steady-state observations where lysozyme showed aggregation tendency at pH 12.2 and also excludes the likelihood of covalent modification promoting oligomerization under this condition.
Steady-state fluorescence anisotropy at short times with 40 µM labeled HEWL
In the presence of 20 mM DTT, there was also a gradual decrease in rss starting from ∼0.09 initially to ∼0.07 after overnight incubation. Thus, in contrast to the control (absence of additive), no increase in rss was observed in the presence of SDS, CTAB and DTT. After observing significant difference in rss of dansyl-labeled HEWL in control and in presence of additives, we proceeded to time-resolved measurements.
Time-resolved fluorescence measurement
Since rss is a function of fluorescence lifetime and the rotational correlation time of the fluorescence probe, we then performed time-resolved fluorescence intensity decay and anisotropy decay of the dansyl-conjugated HEWL to measure the fluorescence lifetime and rotational correlation times of dansyl probe, respectively, under experimental conditions. Average fluorescence lifetime of dansyl labeled HEWL at pH 7 and pH ns) was consistent with earlier reported values (Homchaudhuri et al., 2006), but the appearance of three lifetime components reflects the structural heterogeneity of lysozyme in solution (Wang et al. The changes) in fluorescence lifetime of dansyl probe in presence of CTAB may arise from binding and subsequent electrostatic interaction between positively charged CTAB with negatively charged HEWL at pH 12.2 (Chatterjee et al., 2002).
Time-resolved anisotropy decay measurements
Analysis of the anisotropy decay profile revealed the presence of two correlation times at 30, 360 and 1500 minutes of incubation (Table 5.3, entries 3-5). The anisotropy decay data indicate an r∞ value of ~0.04 after 30 minutes, which decreased to ~0.02 after 360 minutes and remained almost constant after 1500 minutes of incubation. Thus, it is clear from Figure 5.4 and Table 5.3 that the dansyl probe rotational correlation times, obtained from anisotropy decay, serve as an excellent indicator of the process of HEWL aggregation.
Steady-state fluorescence anisotropy at longer time duration
This value was constant between for several weeks thereafter and reflects a monomeric similar state as observed with dansylated HEWL at pH 7. From these observations it is clear that all the additives used are effective in inhibiting the growth of HEWL aggregates over several weeks. to stop While the effect of surfactants, SDS and CTAB is similar to earlier times, the dramatic influence of DTT to stop aggregation is notable and also seems potent enough for long periods of time.
Circular Dichroism spectroscopy
The ~7 ns and ~10 ns rotational correlation times observed in the aggregates in the presence of DTT and CTAB, respectively, together with their low rss after 720 h, reflect the relatively more developed structures observed in CD compared to pH 12.2 alone or in the presence SDS. In contrast to the signature of amyloid, which shows a cross β-sheet, we could not observe these features in the CD spectra of HEWL at pH 12.2 (Figure 5.10). It is possible that amyloid fibrils represent a minor fraction in the mixture of globular aggregates and amyloid fibrils that exist at pH 12.2 after several weeks of incubation.
ANS fluorescence
It is clear that in the presence of DTT, hydrophobic regions of HEWL are not exposed at alkaline condition, thus limiting their interactions leading to aggregation. Curves from top to bottom at different time points are based on emission maximum at 460 nm.
Thioflavin T fluorescence
The curves from top to bottom at different time points are based on the emission maximum at 550 nm.
Estimation of free thiol groups
To establish the importance of late disulfide bond formation in aggregates, we added 20 mM DTT to HEWL samples at pH 12.2 after 120 h and observed ThT fluorescence in control and DTT samples. Observation of Figure 5.14B indicates that at least one thiol of each HEWL monomer may be involved in fresh disulfide bonds after three days, making it likely that they are intermolecular in nature. Once these disulfide bonds are formed, aggregates are tightly packed and assembled, preventing DTT or DTNP or OH- from gaining any access to the inner core of the aggregate (Figures 5.15 and 5.14B).
HEWL activity
This observation was consistent with our previous steady state data where irreversibility in lysozyme accumulation was observed after 60 min (Homchaudhuri et al., 2006) of incubation. The gradual decrease in catalytic activity accompanied by a simultaneous increase in the steady-state anisotropy of lysozyme at pH 12.2, as shown in Figure 5.16, indicates the increase in the total population with the decrease in the native HEWL population. In the presence of additives, Figure 5.16 shows that except with 14 mM SDS at 30 min, none of the additives could help recover any activity even after 1 h.
HEWL proteolysis
Initially, the lower recovery of HEWL activity by SDS indicates weakly bound protein surfactants, which was consistent with the fast dansyl dynamics (~9.4 ns) observed at 30 min of incubation (Table 5.3, point 6), whereas in the presence of CTAB, HEWL was tightly bound (~14 ns, Table 5.3, point 9) to oppositely charged surfactants via electrostatic interaction. Thus, Tricine-SDS PAGE together with absorbance data 210 shows minimal hydrolysis of HEWL in the above mentioned samples. The amount of HEWL loaded in each well is 29 µg, except for HEWL with CTAB, which had 19 µg of protein.
Mechanism
The interaction between HEWL at pH 12.2 with anionic surfactant SDS and oppositely charged surfactant CTAB leads to the detachment of the aggregates at alkaline pH. In light of observations where disulfide bonding is observed to stabilize the native structure of β2-microglobulin and thereby inhibit its fibrillization (Smith and Radford 2001), we suggest from our findings that reduction of disulfide bonding using DTT enhances the growth of HEWL aggregates at pH hindered. 12.2, if applied from the beginning. The proposed mechanism for aggregation of HEWL at pH 12.2 in the presence and absence of SDS, CTAB and DTT is shown schematically.
Preincubation of chitotriose with lysozyme: An alternative approach to reduce aggregation propensity of hen egg white lysozyme
- Binding of chitotriose with HEWL
- ANS fluorescence
- Thioflavin T fluorescence
- SDS PAGE
- HEWL activity
The enhanced fluorescence of ANS by HEWL at pH 12.2 was consistent with the previous observation described in Chapter 5. We used thioflavin T fluorescence to monitor HEWL amyloid in pH 12.2 incubated protein under different experimental conditions. Enzymatic activity of HEWL incubated at pH 12.2 in the presence of inhibitors is another tool to quantify inhibition of aggregation.
Enhanced thioflavin T fluorescence selectively inside anionic micelles and mammalian cells
Absorption of thioflavin T with surfactants
Unlike Figure 7.2, where a dramatic change in ThT absorption was observed with SDS, no significant change in ThT absorption spectrum was observed in the presence of CTAB or Triton 20 to 200 μM (Figure 7.4) revealed a gradual decrease in ThT absorption at 412 nm. Compared to anionic surfactant SDS, the absence of any shift in the absorption spectrum of ThT in the presence of cationic surfactant CTAB or neutral surfactant Triton X-100/Tween 20 (Figure 7.3) suggests that ThT does not enter these micelles.
Fluorescence emission of thioflavin T with surfactants
We next sought to determine the reasons behind the ∼13-fold increase in ThT fluorescence in the presence of SDS. Using Rhodamine 101 as a quantum yield standard, we calculated nearly threefold increase in quantum yield of ThT in the presence of 5 mM SDS. Thus, a nine-fold increase in the fluorescence of ThT in the presence of SDS is roughly predicted, while the observed increase was about thirteen-fold.
Eukaryotic cells imaging
Inside the cell, it is likely to encounter and bind to a wide variety of components such as nucleic acids, membranes and proteins, resulting in fluorescence. The negative charge on the phosphate of the polar phospholipid headgroup in the membrane is likely to facilitate the entry of ThT into the cell based on our results with SDS. However, the mechanism of ThT entry into the cell is currently not clear.
Concluding remarks
Summary
Tryptophan fluorescence can be used as a probe to locate residual structures in denatured proteins using fluorescence anisotropy and quenching by external quencher. The anionic surfactant SDS, the cationic surfactant CTAB, the disulfide-cleaving agent DTT, and the competitive inhibitor chitotriose effectively inhibit the aggregation of lysozyme under alkaline conditions. ThT can be effectively used to monitor micellization of anionic surfactant and used as a membrane-permeant dye to fluorescently label mammalian cells.
Scope of future works
These are the potential works that can be explored based on the current work. The cytotoxic effect of soluble HEWL oligomers can be studied to monitor the pathways leading to cell death. As a new fluorescent dye, ThT can be tried to monitor the growth kinetics of prokaryotic and eukaryotic cells.
Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Formation of amyloid aggregates from human lysozyme and its disease-associated variants using hydrostatic pressure. Chiral bias of amyloid fibrils revealed by the twisted conformation of Thioflavin T: an induced circular dichroism/DFT study.
List of publications
Satish Kumar and Rajaram Swaminathan. Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures
