Chapter 7: Thesis summary and future perspectives
4.2 Results and Discussion
4.2.1 Steady state fluorescence intensity and fluorescence anisotropy studies of PEST M1 tryptophan
To investigate the effect of pH on structure and dynamics of PEST M1, intrinsic fluorescence of its single tryptophan70 (residue 70, present at C-terminus) was monitored.
Fluorescence emission and steady state fluorescence anisotropy (rss) of PEST M1 tryptophan was recorded at different pH (3-11), to extract the information about environment around indole ring and its rotational dynamics at various pH. Fluorescence
emission spectra of PEST M1 at different pH (3-11) were collected by exciting the samples at 295 nm. Figure 4.2.1A shows the emission spectra of PEST M1 tryptophan and its derivative known as NATA (N-acetyl tryptophan amide) at different pH (3-11) in presence of 25 mM NaCl. The integrated fluorescence intensity of PEST M1 tryptophan decreases gradually from alkaline pH (11) to the acidic pH (3) (Figure 4.4.1B). The fluorescence emission spectra of PEST M1 tryptophan shows a progressive blue shift in its emission maxima (347 nm to 341 nm) from pH 11 to pH 3 (Figure 4.2.1C). The observed fluorescence emission spectra of NATA display negligible change at different pH (3-11) as it is fully exposed to solvent. The decrease in the tryptophan fluorescence intensity and blue shift in its emission maxima hints that tryptophan of PEST M1 is buried inside the protein core at acidic pH. Figure 4.2.1D depicts the steady state anisotropy (rss) of PEST M1 tryptophan and NATA at different pH (3-11) in presence of 25 mM NaCl. Substantial increase was observed in the rss of PEST M1 tryptophan when moving from pH 11 to pH 3.
Change in the anisotropy of NATA was insignificant at different pH (3-11). An increase in the anisotropy at acidic pH may be due to hindrance of tryptophan rotation as it buried inside the protein at pH 3. Blue shift in the emission maxima and increase in the rss value indicates local folding of PEST M1 at acidic pH in the vicinity of tryptophan.
Next, we investigated the effect of pH on structure of PEST M1 in presence of excess salt (250 mM NaCl). Excess salt was used to study involvement of electrostatic interaction in the folding of PEST at lower pH. The charge localized on the side chains of amino acids will be stabilized by introducing excess salt counter ions and could influence the folding of PEST in acidic pH. Steady state fluorescence and anisotropy of PEST M1 tryptophan was monitored to study the effect of pH on its structure and dynamics in presence of excess salt.
Figure 4.2.1E, F, G and H depicts the fluorescence spectra, integrated fluorescence yield, fluorescence emission maxima and steady state anisotropy of PEST M1 tryptophan and NATA at different pH (3-11) in presence of 250 mM NaCl. The changes observed in the fluorescence spectra, emission maxima and anisotropy of PEST M1 in presence of 250 mM NaCl at different pH (3-11) was similar to the changes observed in presence of 25 mM NaCl. This similar but not identical change in fluorescence spectra and anisotropy of PEST M1 in presence of 250 mM NaCl indicates the involvement of interactions aside from electrostatics in the folding of PEST at acidic pH.
Figure 4.2.1: Effect of pH on steady state fluorescence and fluorescence anisotropy of 20 μM PEST M1 and 20 μM NATA dissolved in different 25 mM buffers (pH 3-11), containing 250 mM NaCl and 5 mM TCEP. [E] Steady state fluorescence spectra; [F] integrated fluorescence yield;
[G] fluorescence emission maxima and [H] steady state anisotropy of PEST M1 tryptophan and NATA at different pH.
Further, the fluorescence spectra and anisotropy of PEST M1 tryptophan was monitored in presence of 6 M Gdn.HCl, to completely unfold the PEST fragment at different pH (3-11).
Figure 4.2.1I, J, K and L represent the fluorescence spectra, integrated fluorescence yield, fluorescence emission maxima and fluorescence anisotropy of PEST M1 tryptophan and NATA at various pH (3-11) in presence of 6 M Gdn.HCl. In presence of 6 M Gdn.HCl, decrease in fluorescence intensity was minor and change in emission maxima was insignificant at different pH (3-11) as compared with these changes observed in presence of 25 and 250 mM NaCl. The emission λmax of tryptophan ranges from 346-347 nm in comparison to ~343 nm at high salt and ~341 nm at low salt, clearly indicating a more exposed indole ring in presence of 6 M Gdn.HCl. The change in anisotropy values was
Figure 4.2.1: Effect of pH on steady state fluorescence and anisotropy of 20 μM PEST M1 and 20 μM NATA dissolved in different 25 mM buffers (pH 3-11), containing 25 mM NaCl, 6 M Gdn.HCl and 5 mM TCEP. [I] Steady state fluorescence spectra; [J] integrated fluorescence yield;
[K] fluorescence emission maxima and [L] steady state fluorescence anisotropy of PEST M1 tryptophan and NATA at different pH.
marginal in presence of 6 M Gdn.HCl suggesting no major structural alteration occurs in PEST M1 as its tryptophan freely rotates at various pH (3-11) (Figure 4.2.1L). These observations clearly indicate a predominantly unfolded structure of PEST M1 at different pH (3-11) in presence of 6 M Gdn.HCl.
The rss value depends upon fluorescence lifetime (τ) and rotational correlation time (θ) of the protein. So, change in rss may be due to change in lifetime of tryptophan or could arise from change in rotational correlation time (θ) of PEST fragment at different pH (3-11). To resolve actual reason, we monitored lifetime of PEST M1 tryptophan at different pH (3-11).
Figure 4.2.2: Fitted time-resolved fluorescence intensity decay profile of 20 μM PEST M1 tryptophan at different pH (3-11) in presence of [A] 25 mM NaCl and [B] 250 mM NaCl (Left panels). Panels in the right reveal the residual distribution of the fit for each pH condition.