Effect of SDS, CTAB and DTT on the size, dynamics, activity and growth of soluble lysozyme aggregates
5.4 Time-resolved anisotropy decay measurements
µM protein also have same mean lifetime as observed for higher concentrations of proteins. Thus concentration dependent changes in mean lifetime can be ruled out in these cases. 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). This interaction seems to be more pronounced at higher protein concentration where with 80 µM protein, mean lifetime reduces to ~8.5-9.0 ns compared to 40 µM (11-12 ns). Fairly invariant fluorescence lifetime over the period of ~1500 minutes indicates that the rise in rss may not be attributed to change in lifetime however, it was likely that rotational correlation time shall contribute to these changes. The invariance of lifetime data during 0-1500 minutes clearly validates the use of rss to monitor the aggregation of dansyl labeled HEWL. Subsequently we performed time-resolved anisotropy decay measurements to obtain insight into the molecular dynamics of protein in control and in presence of additives.
to a single exponential with a rotational correlation time ~4 ns (see Table 5.3, items 1- 2), accounting for tumbling of the whole protein that is fully monomeric. This value is consistent with previously reported value (3.8 ns) for native lysozyme (Vos et al., 1987). Therefore, the overall 3D structure of the protein in the native state is intact and unchanged during the long duration of incubation. This was in agreement with our earlier results (Homchaudhuri et al., 2006).
Anisotropy decay of 120 µM HEWL in pH 12.2 at different time points is shown in figure 5.4B. While observing r(t) longer time (~30 ns), which we termed as residual anisotropy (r∞) there was small but significant r∞ (~0.03) at 30 minutes. As time progresses, this value rises significantly (>0.10) at 360 minutes and remains constant till next day (1500 minutes). Analysis of anisotropy decay profile revealed the presence of two correlation times at 30, 360 and 1500 minutes of incubation (Table 5.3, items 3-5). Looking at the fast rotational component, a value of ~2.4 ns (amplitude of 0.44) was observed at 30 minutes. Subsequently at 360 minutes this component is slowed down (~5 ns) and it remains fairly slow (~5 ns) thereafter at 1500 minutes, although a minor increase in the amplitude of this component (0.26 to 0.38) was seen after overnight incubation. Thus, changes in fast component which indicates the segmental motion of dansyl probe (attached to ε-amino group of L-lysine residue in lysozyme) indicates that probe that was initially fast and free, became slow and hindered later on as time progresses. While observing the slow component which reflects the global motion of protein, correlation time of ~10 ns with amplitude 0.56 was obtained after 30 minutes of incubation. This ~10 ns component may arise from the global tumbling of a small oligomeric HEWL or from an extended topology of a misfolded monomer. After 360 minutes this component increased significantly to ~73 ns (0.74) and ~60 ns (0.62) after 1500 minutes, indicating the formation of large multimeric HEWL aggregates and their reorganization after overnight incubation. So, six hours exposure to alkaline pH appears sufficient to trigger formation of large aggregates in HEWL. We used this condition as a control for different manoeuvres that follow to inhibit the process of aggregation.
Figure 5.4C shows the effect of 14 mM SDS on HEWL aggregation at pH 12.2. The anisotropy decay data indicates r∞ value of ~0.04 after 30 minutes which decreased to ~0.02 after 360 minutes and remain almost constant after 1500 minutes of incubation. The details of the fits obtained from these traces are displayed in Table 5.3 (items 6-8). After 30 minutes fast segmental motion of 2.1 ns was observed with
an amplitude of 0.29. This value was slightly increased to 2.4 ns at 360 minutes and 2.8 ns at 1500 minutes with higher amplitude (0.55-0.58). This reflects the fairly unrestricted segmental mobility of dansyl group at later times. While looking to slow component, we observed 9.4 ns (0.71) correlation time at 30 minutes which was similar to that observed with control at same time point. At 360 and 1500 minutes, this value was constant with ~14 ns (0.45-42), indicating an oligomeric HEWL with loose molecular packing perhaps with bound SDS micelles. These observations clearly indicate that SDS inhibits progress of HEWL aggregation at pH 12.2.
Figure 5.4D depicts the influence of 3 mM CTAB on the kinetics of HEWL aggregation at alkaline condition. Residual anisotropy at 30 minutes shows the value of ~0.05 which was reduced to ~0.03-0.02 at 360 and 1500 minutes respectively.
Fitted parameters for each of the traces are shown in Table 5.3 (items 9-11). Fast component of ~2.2 ns with 0.48 amplitude was monitored after 30 minutes, which showed faster segmental motion of ~1.5 ns with amplitude of 0.45 after 360 minutes and remain almost constant thereafter upto 1500 minutes of observations. Slow component also exhibited decreasing trends with ~14 ns at 30 minutes to ~12 ns after 360 minutes then ~10 ns after 1500 minutes of incubation with marginal increase in amplitude. These results suggest that HEWL exists as a loosely packed oligomer, whose aggregation propensity is diminished in prolonged presence of CTAB.
To rule out any contribution of solution viscosity for slow overall tumbling (10-14 ns) of the protein in presence of 14 mM SDS or 3 mM CTAB, we performed bulk viscosity measurements using Ostwald’s viscometer and observed no significant change in solution viscosity in presence of these surfactants.
Figure 5.4E shows the influence of 20 mM DTT on the growth kinetics of lysozyme aggregates in solution at pH 12.2. Residual anisotropy of <0.05 at 30 ns after 30 minutes of incubation indicates fast anisotropy decay. This value was exhibiting a decreasing trend at 360 and 1400 minutes. Table 5.3 (items 12-14) display the details of the fits obtained by these traces. Fast component of ~1.2 ns was observed after 30 minutes of incubation, which increased marginally to ~1.9 ns at 360 minutes and 1.4 at 1400 minutes. Amplitude of fast motion was also raised from 0.45 to 0.64 and finally to 0.81. This observation indicates increasing freedom for segmental motion in presence of DTT. Slow component between 7-8 ns with concomitant decrease in amplitude (0.55 to 0.19) was observed for same time span indicating the elongated monomeric form of HEWL. These observations indicate that
inhibitory effect of DTT is emphatic on HEWL oligomerization, compared to SDS and CTAB.
In order to investigate the influence of low monomer concentration on dynamics of the aggregates, we carried out experiments with 20 µM lysozyme at pH 12.2. Figure 5.4F reveals the anisotropy decays observed at 30, 360 and 1500 minutes of exposure to alkaline pH. At 30 minutes of incubation, r∞ was nearly zero which marginally increased to ~0.02 after 1500 minutes reflecting the slow progress of aggregation. The decay parameters are listed in Table 5.3, items 15-17.
Compared to higher monomer concentration (120 µM protein), the amplitude for the fast segmental motion (0.55) at 30 minutes is significantly higher indicating a relatively unhindered mobility. As time progresses, this rotational component becomes noticeably faster (~1.3 ns) retaining significant amplitude quite unlike the ~5 ns component at higher monomer concentration where segmental motion is slowed down and restricted as time progresses. The slow component shows a value around 11-12 ns at 360 minutes and later, indicating a small oligomer in contrast to the large multimeric aggregate (~60 ns) observed at higher monomer concentration. It is thus, apparent that a low monomer concentration has a critical impact on the aggregation growth kinetics as would be expected for a concentration dependent phenomenon.
It is thus clear from Figure 5.4 and Table 5.3 that the rotational correlation times of dansyl probe obtained from anisotropy decays serve as an excellent indicator of the process of HEWL aggregation. The reduced rotational correlation times in presence of 14 mM SDS, 3 mM CTAB and 20 mM DTT compared to pH 12.2 control clearly indicates the effectiveness of these molecules in arresting the growth of HEWL aggregates at alkaline pH.
The figures obtained during the analyzing the anisotropy decays are shown in Figures 5.5 to 5.8.
Table 5.3 Decay parameters recovered from fits to anisotropy decay curves in Figure 5.4
ainitial anisotropy; bsteady-state anisotropy calculated from fit; crotational correlation time(s); dfractional amplitudes associated with correlation time; ereduced chisquare for the fit. The errors in the values reported for φ1 are within 10%, while those for φ2
are 5%, based on results from multiple experiments.
Condition t
mins
r
0ar
ssbφ
1 c(ns)
φ
2c(ns)
α
1 dα
2dχ
2e1 pH 7 30 0.18 0.04 4.1 -- 1.0 -- 1.5
2 pH 7 1600 0.18 0.05 4.0 -- 1.0 -- 1.5
3 pH 12.2 30 0.29 0.10 2.4 10 0.44 0.56 1.2
4 pH 12.2 360 0.29 0.20 4.9 73 0.26 0.74 1.5
5 pH 12.2 1500 0.27 0.17 4.8 58 0.38 0.62 1.4
6 pH 12.2, 14 mM SDS 30 0.25 0.10 2.1 9.4 0.29 0.71 1.3 7 pH 12.2, 14 mM SDS 360 0.23 0.09 2.4 14 0.55 0.45 1.3 8 pH 12.2, 14 mM SDS 1500 0.23 0.08 2.8 14 0.58 0.42 1.5 9 pH 12.2, 3 mM CTAB 30 0.27 0.14 2.2 14 0.48 0.52 1.1 10 pH 12.2, 3 mM CTAB 360 0.29 0.12 1.5 12 0.45 0.55 1.3 11 pH 12.2, 3 mM CTAB 1500 0.29 0.10 1.3 10 0.51 0.49 1.2 12 pH 12.2, 20 mM DTT 30 0.26 0.08 1.2 7.5 0.45 0.55 1.4 13 pH 12.2, 20 mM DTT 360 0.20 0.05 1.9 8.4 0.64 0.36 1.4 14 pH 12.2, 20 mM DTT 1400 0.24 0.05 1.4 7.1 0.81 0.19 1.6 15 pH 12.2, 20 µM 30 0.21 0.06 2.4 8.9 0.55 0.45 1.4 16 pH 12.2, 20 µM 360 0.25 0.07 1.3 11 0.49 0.51 1.2 17 pH 12.2, 20 µM 1500 0.28 0.08 1.4 12 0.57 0.43 1.9
log (counts)
1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5
time (ns)
10 20 30 40 50
r (t)
-0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25
residuals
-5 0 5
time (ns)
10 20 30 40 50
residuals -5
0 5
Intensity Decay Anisotropy Decay
Initial anisotropy = 0.27 Rot. Corr. times are 4.8, 58.6 ns Amplitudes are 0.38, 0.62 Steady state anisotropy = 0.17 Anisotropy Decay of 120 M dansyl labeled lysozyme in pH 12.2 buffer at t = 1500 minutes
Figure 5.5 The plots shown above correspond to (i) IRF (blue); (ii) measured Ipar
(black); (iii) fitted I
par(pink); (iv) measured I
per(black); (v) fitted I
per(red). The
measured anisotropy decay is shown in dark green and fitted anisotropy decay is
depicted by blue curve. The corresponding residuals are shown in black curves below
the intensity decay plots. Upper residual represents for parallel intensity decay (I
par)
while lower residual represents for perpendicular intensity decay (I
per).
Figure 5.6 The details of curves are same as depicted in Figure 5.5.
log (Counts)
1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5
time (ns)
10 20 30 40 50
r (t)
-0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25
χ2 = 1.5
Initial anisotropy = 0.23 Rot. corr. times are 2.8, 14.4 ns Amplitudes are 0.58, 0.42 Steady state anisotropy = 0.08
Intensity Decay Anisotropy Decay
Anisotropy Decay of 120 µM dansyl labeled lysozyme in pH 12.2 buffer containing 14 mM SDS at t = 1500 minutes
residuals -5
0 5
time (ns)
10 20 30 40 50
residuals -5
0 5
Figure 5.7 The details of curves are same as depicted in Figure 5.5.
log (counts)
1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5
time (ns)
10 20 30 40
r (t)
-0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25
residualsresiduals -5 0 5
times (ns)
10 20 30 40
-5 0 5
χ2 = 1.2
Initial anisotropy = 0.29 Rot. corr. times are 1.3, 10.1 ns Amplitudes are 0.51, 0.49 Steady state anisotropy = 0.10
Intensity Decay Anisotropy Decay
Anisotropy Decay of 80 µM dansyl labeled lysozyme in pH 12.2 buffer containing 3 mM CTAB at t = 1500 minutes
Figure 5.8 The details of curves are same as depicted in Figure 5.5.
1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5
time (ns)
10 20 30 40 50
r (t)
-0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25
log (counts)
times (ns)
10 20 30 40 50
residuals
-5 0 5
residuals
-5 0 5
χ2 = 1.6
Initial anisotropy = 0.24 Rot. corr. times are 1.4, 7.1 ns Amplitudes are 0.81, 0.19 Steady state anisotropy = 0.05
Intensity Decay Anisotropy Decay
Anisotropy Decay of 120 µM dansyl labeled lysozyme in pH 12.2 buffer containing 20 mM DTT at t = 1400 minutes