This can lower the ProCharTS absorption and autoluminescence from charge transfer states. Taken together, charged amino acids and charged amino acid-rich proteins indicate that ProCharTS and associated charge transfer states are emissive in nature.
Introduction and Literature Review
Experimental Techniques and Materials & Methods
Intrinsic luminescence from charged, non-aromatic amino acids and charged monomeric proteins
Human Serum Albumin (HuSA): A model protein to investigate ProCharTS
Effect of ProCharTS on extrinsic and intrinsic fluorescent probes in proteins
ProCharTS: A label-free approach to detect aggregation of hen egg white lysozyme (HEWL)
Thesis Summary and Future Perspectives
Introduction
Charge Transfer
Electron transfer reactions between a donor (D) and an acceptor (A) are mostly represented by the following scheme. The term A in equation 1.2 depends on the nature of the electron transfer reaction (for example, bimolecular or intramolecular), kB is the Boltzmann constant, T is the temperature, ∆𝐺0 is the standard free energy of the reaction, and λ is the reorganization term consisting of the solvation (λs) and vibrational (λv) components (equation 1.4).
Charge transfer in biological systems
- Charge transfer deriving the photosynthesis
- Electron transfer and the respiratory chain
- Charge transfer in nucleic acids
- Charge transfer in peptides and proteins
- Protein Charge Transfer Spectra (ProCharTS)
This electron transfer process occurs within the pigment-protein complex called the reaction center (RC) (Deisenhofer et al 1985). This has also motivated the construction of electron transfer based biosensors (Ghindilis et al 1997) and electrochemical immunological biosensors (Saen-Oon et al 2013).
Experimental techniques
- Spectroscopy
- Absorption Spectroscopy
- Basics of UV-Visible spectroscopy
- Fluorescence Spectroscopy
- Steady-state fluorescence
- Steady-state fluorescence anisotropy
- Time-resolved fluorescence
- Analysis of time-resolved fluorescence intensity decay
- Fluorescence quenching
The decay of the excited state population is exponential in nature and is represented by Eq. The calculated average lifetime of the fluorophore is proportional to the area under the intensity decay curve.
Materials
In the Stern-Volmer plot, kq, bimolecular quenching rate constant describes the efficiency of quenching. For a diffusion-controlled quenching, the value of kq around 1x1010 M-1s-1 shows the maximum possible value of kq in the solution.
Methods
- Estimation of protein concentration
- UV-Visible absorbance measurements
- Steady-state fluorescence measurements
- Steady-state anisotropy measurements
- Stokes shift measurements
- Quantum yield measurements
- Time-resolved fluorescence measurements
- Analysis of time-resolved fluorescence data
- Protein unfolding
- Labelling of protein with Dansyl Chloride
- Aggregation of HEWL
- Aggregation at alkaline pH
- Aggregation at pH 5.0
- Aggregation at pH 2.0
- Inhibition of HEWL aggregation
- Thioflavin T Assay
All samples (proteins and amino acids) were freshly prepared in deionized water and all measurements were performed at 25 °C using quartz cuvettes with a path length of 10 mm (Hellma; Z600210). Luminescence emission spectra for all proteins and amino acids (Lysine·HCl, Glutamate and Lysine) were collected at different excitation wavelengths and 410 nm with a slit width of 2 nm for excitation and 15 nm for emission. All samples were buffered at pH 7.0 (50 mM phosphate buffer) to avoid any effect of a mere change in pH.
Similarly, luminescence spectra were measured in deionized water for all proteins and amino acids while for 9,10-DPA (Diphenylanthracene) the measurement was made in cyclohexane (99%). All samples including the standard were excited at 355 nm with a slit width of 1 nm and almost the complete emission spectrum was collected between 370 - 690 nm with a slit width of 5 nm. Integrated luminescence (area under the full emission spectrum) was calculated between 370–690 nm for all samples for quantum yield calculation.
Introduction
Much work has been done so far (Homchaudhuri & Swaminathan 2001, Homchaudhuri & Swaminathan 2004, Prasad et al 2017) on absorption arising from charged amino acids. ProCharTS among charged amino acids were also found to depend on the pH and ionic strength of the solvent. Among all charged amino acids, lysine has the highest extinction coefficient (ɛ) at 270 nm, followed by aspartate and glutamate salts (Prasad et al 2017).
Here in this chapter, the luminescence arising from charge transfer states between various charged, non-aromatic amino acids (Lysine, Lysine∙HCl and Glutamate) is presented and discussed. Amino acids with a capped amino/carboxy terminal are also studied in parallel to monitor the role of charge on N/C terminals in charge transfer transitions. In addition to amino acids, several proteins rich in charged amino acid residues are also examined for their likely luminescence in their monomeric form.
Results and Discussions
- ProCharTS observed among Lysine, Lysine∙HCl and Glutamate
- ProCharTS observed among amino acids are emissive in nature
- Luminescence from charge transfer states in amino acids mirrors the change in ProCharTS absorbance
- Effect of addition of equimolar concentrations of Lysine∙HCl and Glutamate
- Selection of different proteins rich in charged residues
- Absorbance and extinction coefficient of different proteins and amino acids
- ProCharTS observed from monomeric proteins
- Emission spectra of different proteins and amino acids
- Excitation spectra of different proteins and amino acids
- Stokes shift
- Variation in luminescence yield with excitation wavelength
- Quantum yield of different proteins and amino acids at 355 nm
- Linearity in luminescence at λ ex 355 nm
- Relationship between the luminescence and product of ɛ and Φ
- Luminescence lifetime of protein charge transfer states .1 Luminescence lifetime at λ ex 340 nm
- Luminescence lifetime at λ ex 295 nm
The luminescence yield of all amino acids and proteins was determined at different excitation wavelengths (Figure 3.11). The value of this parameter (ɛ355 x Φ355) for different proteins and amino acids is presented in table 3.2. Decomposition of the luminescence intensity of all proteins and amino acids at λex 340 nm are shown in Figure 3.16 along with their fitted and fitted residues.
The individual components and the average lifetime of all proteins and amino acids are shown in Table 3.3. For λex 295, the luminescence intensity decay profile of all amino acids and proteins (except Trp-containing proteins, PEST M1, and HuSA) is presented in Figure 3.20. The individual components and the average lifetime of all proteins and amino acids are shown in Table 3.4.
Conclusions
Introduction
And this is the motive of the current chapter to illustrate the effects of the above conditions on HuSA's ProCharTS. To achieve this, absorption-based studies of HuSA were performed to reveal how the change in pH and ionic strength can alter ProCharTS. In addition to absorption-based studies, luminescence from charge-transfer states is also investigated to show how the luminescence from charge-transfer transitions can uniquely confirm and interpret the above results.
In the later part of the chapter, intrinsic luminescence from charge transfer states of HuSA molecules is presented. Since, there have been reports on the origin of intrinsic fluorescence from oligomeric HuSA molecules (Bhattacharya et al 2017, Bhattacharya et al 2014); luminescence from HuSA was investigated in more detail. To establish that the observed luminescence originates from monomeric HuSA molecules and not from some type of aggregated or oligomeric entity, steady-state anisotropy measurements were performed using Dansyl-labeled HuSA.
Results and Discussions
- Effect of pH and ionic strength on ProCharTS of HuSA
- Prevalence of luminescence from charge transfer states in HuSA
- Intrinsic luminescence observed from monomeric HuSA
- Unfolding of HuSA decreases ProCharTS
Fluorescence intensity of Dansyl-HuSA and the mixture of Dansyl-HuSA and free unlabeled HuSA are shown in Figure 4.5A. The change in spectral shape (increasing FWHM with increasing concentration of unlabeled HuSA) in Figure 4.5B is also evidence for this. Second, large red shift in the fluorescence spectrum of the buried tryptophan residues (Eftink 1994, Ervin et al 2000) was observed (Figure 4.8), is evidence of HuSA unfolding.
As observed in Figure 4.7, the unfolding caused by Gdn∙HCl resulted in the decrease of ProCharTS in HuSA. The goodness of fit can be observed from the randomness of the fitted residuals as shown in Figure 4.10B. The fitted parameters for luminescence intensity decrease of HuSA in the presence of 0, 3 and 6 M Gdn∙HCl are shown in Table 4.1 and Figure 4.11.
Conclusions
Introduction
Finally, the effects of charged residues present in the protein (without aromatic amino acids) on the fluorescence lifetime of Trp were studied. The origin of the multiple lifetime of Trp is assumed to be mainly due to emission from two almost identical electronic absorption transitions (state 1La and 1Lb) of Trp (Valeur & . Weber 1977) or due to the presence of rotameric structures around Cα ‒ Cβ bonds (Ghisaidoobe & Chung 2014, Pan & Barkley 2004). This study may provide an alternative insight into the origin of the highly exponential decay of Trp in proteins, depending on contributions from charged residues in proteins.
On the other hand, extrinsic fluorophore Dansyl is investigated for changes in several photophysical characteristics, mainly its steady-state fluorescence and its fluorescence lifetime when labeled to a protein (HuSA) rich in charged amino acids and in excess presence of such charged proteins. As shown in Chapter 4, the change in spectral shape and intensity of Dansyl-HuSA at λex 340 nm in the presence of unlabeled HuSA is further investigated in this chapter to evaluate any associated changes in fluorescence lifetime. These studies can help in understanding the effects of charged residues on both the extrinsic and intrinsic protein fluorophores, and provide a clue to the energy transfer phenomenon with ProCharTS as an acceptor and the presence of an additional chromophore such as ProCharTS in the protein.
Results and Discussions
- Effect of ProCharTS on fluorescence of an extrinsic probe, Dansyl
- Effect of ProCharTS on the fluorescence of intrinsic probe
- Effect of Lysine on the fluorescence of NATA
- Effect of Lysine∙HCl on the fluorescence of NATA
- Effect of Glutamate on the fluorescence of NATA
- Effect of charged residues in protein on the fluorescence of NATA
- Effect of inserted Trp on the ProCharTS luminescence of PEST wt
As observed from Figure 5.2, there is a regular increase in the FWHM (full width at half maximum) of the normalized fluorescence spectrum of Dansyl-HuSA in the presence of increasing concentration of unlabeled HuSA. The steady-state fluorescence of NATA at λex 295 nm was observed to decrease in the presence of Lysine (Figure 5.7A). Steady-state fluorescence shows a similar decrease in NATA fluorescence intensity with increasing concentration of Lysine∙HCl (Figure 5.12A and irregular bars in Figure 5.12B).
Time resolved fluorescence studies for the above samples followed a similar 2 exponential decay (Figure 5.19) as observed in the above sections in the presence of Lysine and Lysine∙HCl. This indicates the marginal decrease in the mean lifetime of NATA in the presence of given concentrations of glutamate (Figure 5.20). The fluorescence intensity decay of both proteins at λex 295 nm reveals a three exponential decay (Figure 5.25 and Table 5.9).
Conclusions
Introduction
In addition, the sensitivity of ProCharTS in detecting protein aggregates was compared with THT assays. Several groups have reported intrinsic fluorescence arising from oligomeric (Bhattacharya et al 2017, Bhattacharya et al 2014) as well as protein amyloid fibrils (Chan et al 2013, del Mercato et al 2007, Tikhonova et al 2018). Such fluorescence from protein oligomers and protein fibrils is called intrinsic fluorescence (Chan et al 2013, del Mercato et al 2007, Pinotsi et al 2013) or intrinsic dark blue/blue fluorescence (Bhattacharya et al 2017, Tikhonova et al 2018).
Here in this chapter, the luminescence of the HEWL aggregates is observed and evaluated as charge transfer state emissions from ProCharTS. Apart from this, the quantum yield and luminescence lifetime of HEWL oligomers and fibrils formed under acidic conditions are also reported. Taken together, the detection of protein aggregates based on protein charge transfer spectra (ProCharTS) and a simpler way to distinguish between the oligomers and fibrils, based on the shift in the luminescence spectra at λex.
Results and Discussions
- Detection of HEWL aggregates by ProCharTS
- ProCharTS of HEWL aggregates formed at alkaline pH
- ProCharTS of HEWL aggregates formed at acidic pH
- Intrinsic Luminescence from HEWL aggregates
The increase in ProCharTS was observed with increasing age of aggregates formed at pH 5 or pH 2 (Figure 6.3AB). As a result, the ProCharTS observed for aggregates at pH 2 are higher than those at pH 5 (Figure 6.3AB). The aggregates formed at pH 5 show a continuous increase in the ProCharTS (Figure 6.4B) while the ThT fluorescence remained the same (Figure 6.3D).
By prolonging the incubation period, the intensity of luminescence of the aggregates increased both for pH (Figure 6.6AB). THT fluorescence for aggregates formed at pH 2 increases concomitantly with time, which is reflected in a gradual increase in luminescence intensity (Figure 6.6 AC). For comparison, the decrease in luminescence intensity of HEWL monomers and 8-day-old aggregates formed at one of the pHs is considered and shown in Figure 6.11.
Conclusions
Thesis Summary
Future Directions
Appendix-I
Appendix-II
All excitation was performed with a slit width of 2 nm and emission was collected with a slit width of 15 nm. Luminescence spectra were collected for Aex 355 nm between 370-690 nm using an excitation slit width of 1 nm and an emission slit width of 5 nm.
Appendix-III
Panel A shows the distribution, while panel B shows the residuals obtained from the MEM fit.
Appendix-IV
List of Publications
List of conference papers
List of national conferences attended
Introduction to Electron Transfer in Inorganic, Organic and Biological Systems in Electron Transfer in Inorganic, Organic and Biological Systems, pp. Effect of the electric field generated by the helix dipole on photoinduced intramolecular electron transfer in dichromophoric α-helical peptides. Conformational effects on the electron transfer efficiency in peptide foldamers based on α,α-disubstituted glycyl residues.
Long-distance proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of the respiratory complex I. Proceedings of the National Academy of Sciences 106: 8537 Lindberg DJ, Wenger A, Sundin E, Wesén E, Westerlund F, Esbjörner EK. Proceedings of the National Academy of Sciences of the United States of America Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K.
