Chapter 4: Steady-state fluorescence and CD measurements of
4.2 Steady-state fluorescence intensity studies of mCM and its mutants 77
To avoid interference by the Tyr residues, fluorescence spectra of mCM proteins were collected after excitation at 295 nm. A fluorescence peak at 340 nm in Wt mCM suggests that the Trp residues are exposed to aqueous medium as shown in Figure 4.2. This is be expected from a protein where the two Trp residues are positioned in a flexible turn connecting two alpha- helices. Absorbance by mCM and its mutants in the 325 nm to 425 nm region is probably due to Protein Charge Transfer Spectra (ProCharTS), which is observed when charged amino/carboxylate groups in the side chains of Lys/Glu act as electronic charge acceptors/donors for photoinduced charge transfer either from/to the polypeptide backbone or to each other (Prasad et al., 2017) (Ansari, Kumar and Ahari, 2018). The single Trp containing mutants displayed a similar profile for the fluorescence
emission spectra (λmax341-342 nm). The fluorescence yield for Wt mCM was noticeably reduced compared to its mutant forms and a slight shift of 1-2 nm in the emission maximum was also recorded. The observed difference in fluorescence intensity between the mutants and wild-type might stem from changes in the environment around the single tryptophan (Trp 26) after replacement of the Trp 24 residue in these mutants or possible interactions between the two tryptophans in close vicinity (viz. energy transfer) exclusively in the Wt mCM.
Figure 4.2. Fluorescence spectra (steady-state) of mCM and its mutants obtained after excitation with 295 nm wavelength (excitation slit: 2 nm, emission slit: 5 nm). 20 μM of each of the proteins were used for the experiment. Fluorescence emission of NATA (10 µM) is shown as dotted curve.
In order to ascertain that the tryptophan fluorescence is not directly affected by the ligand, up to 20-fold excess of the TSA (200 µM) w.r.t the Wt mCM (10 µM) was allowed to bind to the protein and the fluorescence was recorded. Addition of TSA had negligible effect on the tryptophan fluorescence intensity in Wt mCM (Figure 4.3). This indicates that any structural rearrangement in mCM after TSA binding doesn’t alter the flexible turn region or the immediate environment in which the tryptophan(s) resides. A better understanding of the segmental motions and helical compaction after ligand binding was expected from FRET experiments (discussed later). It was also observed that 1mM TSA alone produced fluorescence signals of the order of 103 when excited at 295 nm (Figure 4.3). Hence, all further fluorescence experiments with TSA was done with concentrations much lower than 1mM to limit the background fluorescence from the TSA.
Figure 4.3. Fluorescence spectra of 10 µM Wt mCM (unlabeled with any probe) in presence of 20 µM (1:2) and 200 µM (1:20) TSA.
4.2.1 ANS binding assay of mCM proteins
The TSA binds to the hydrophobic core of mCM ; hence it was quite intended to extract some useful information on the hydrophobic nature of mCM and its mutants in both its free and ligand bound states with the aid of ANS assay (Stryer, 1965). ANS binding to mCM was manifested as a drastic increase in the fluorescence intensity of ANS as shown in Figure 4.4 . ANS is known to bind preferably to molten globular states of proteins (Ptitsyn et al., 1990) and is therefore expected to be a sensitive tool for our studies.
Figure 4.4. (Top) The binding of ANS (40µM) to Wt mCM and all the mutants (20µM). Addition of TSA (40 µM) to mCM (20 µM) before ANS binding affected the binding process negatively. An excitation slit of 2 nm and emission slit of 10 nm was used for the measurements and all samples were excited at 375 nm. (Bottom, left) Integrated fluorescence area and (Bottom, right) emission λmax
wavelengths corresponding to the ANS fluorescence intensities for Wt mCM and its mutants in absence and presence of TSA.
ANS displayed higher fluorescence intensity in Wt mCM compared to Mutant1 and Mutant2. The hydrophobic core/patches of Mutant1 and Mutant 2 could have become more solvent exposed or undergone some structural deviation from the wild 3D configuration.
Surprisingly, Mutant3 reported even higher fluorescence intensity for ANS. An altogether different structural arrangement of the helices in this triple mutant is possible, giving rise to new and additional hydrophobic patches where ANS could bind. Furthermore, TSA was allowed to bind with the protein before incubation with ANS. This brought about a significant reduction in ANS fluorescence in wild as well as all the variants of the protein.
This can happen if TSA binding has rendered ANS inaccessible to its binding sites in the protein. Hence, the TSA and ANS binding sites seem to overlap in the mCM protein. A similar observation was earlier reported by the Hilvert group in ETH Zurich (Vamvaca et al., 2004). This could also mean that a more rigid and tight structure in presence of TSA which closes all entry points for ANS that existed in absence of TSA. Moreover, this study also points to the fact that TSA binds to all forms of mCM, but this binding may not bring about the structural transitions in all forms of mCM necessary for it to carry out its function.
4.2.2 Steady state (ss) FRET measurements
ssFRET data of Wt mCM was recorded to ascertain experimentally the possibility of energy transfer between the tryptophan residues (Trp24/26) in mCM and the dansyl
labeled cysteine residue (Cys 69). A significant decline in the fluorescence intensity of Trp in Wt mCM under native condition, was observed after excitation at 295 nm (Figure 4.5). To confirm that intramolecular FRET is responsible for this decrease in fluorescence, Gdn.HCl (2-6 M) was used to gradually unfold the protein at 25oC and observe its effect on the tryptophan fluorescence. As expected, an increase in tryptophan fluorescence intensity corresponding to a decrease in the FRET efficiency (0.42 to 0.13) is observed in Wt mCM with increasing unfolding induced by Gdn.HCl (Table 4.1). The distance between indoles (donor) of Trp 24 and Trp 26 and dansyl (acceptor) group linked to Cys was calculated to be 23 Å. Since ssFRET is sensitive to sample concentrations, this value is an estimate. Lifetime fluorescence-based FRET measurements for Wt mCM and the mutants were later carried out (please refer Chapter 5) to get more reliable FRET distances (Trp-Cys) for these proteins.
Table 4.1. Change in the intramolecular FRET efficiency of Wt mCM in presence of GdnHcl Wt mCM in GdnHcl FRET efficiency
No GdnHCl added 0.43
2M GdnHCl added 0.33
6M GdnHCl added 0.13
The steady-state FRET efficiency for Wt mCM was calculated using the following formula:
𝐸 = 1 − (𝐹𝐷𝐴⁄𝐹𝐷) 4.1
Where FDA is the fluorescence intensity of tryptophan(donor) in presence of dansyl probe (acceptor) and FD in absence of the acceptor. The λem range taken for calculating FDA and FD was310-400 nm. For FRET efficiency in presence of Gdn.HCl, 20 µM Wt mCM was incubated with 2-6 M Gdn.HCl overnight at room temperature and pH 7.2 before fluorescence measurements were taken.
Figure 4.5. Steady state FRET between Trp24/26 and Cys 69 in Wt mCM. Emission spectra corresponding to both Trp and the dansyl probe are observed after excitation at 295 nm (solid lines). Trp in unlabeled mCM is represented in coloured dashed lines. Addition of GdnHcl gradually increases Trp fluorescence in labeled mCM, whereas the fluorescence of unlabeled mCM remains unaffected in presence of Gdn.HCl.