Electronic absorption of proteins that lack cofactors, prosthetic groups, or noncoded amino acids as part of their structure has traditionally been thought to originate from protein backbone (peptide bond) and aromatic chromophores in the side chains. These side chains are chemical groups attached to the backbone that help the proteins to function. Among the protein chromophores — peptide bond/Trp/Tyr/Phe — the absorption occurs in the 190- to 320-nm region. Wavelengths above 320 nm are believed to be optically silent for such proteins. Such absence of optical sensitivity in the 320- to 800-nm region of the spectrum means that normal proteins in cells or test tubes cannot be tracked or identified using common spectroscopic methods, such as intrinsic UV-VIS absorption or accompanying fluorescence, in a label-free manner. For investigating the structure and function of proteins inside cells, label-free approaches work best. Inserting external probes is tedious and often perturbs the natural state of the protein. An illustration of a chain of amino acids that makes up a protein. Portions of these chains can absorb light and indicate the presence of certain conditions. Courtesy of iStock.com/Christoph Burgstedt. Recent work from the authors and their collaborators has shown that positively and negatively charged polar headgroups of amino acids such as Lys/Arg/His and Asp/Glu/pTyr/pThr/pSer, respectively, in a protein can collectively contribute to significant UV-VIS absorption. This phenomenon arises from protein charge transfer spectra (ProCharTS) spanning the 250- to 800-nm range, with molar extinction coefficients between 7000 and 500 M−1 cm−1. Importantly, the intensity of this absorption, originating from photoinduced electron transfer, is sensitive to the spatial distance separating the charged atoms in the folded protein, aside from the charge itself. This makes ProCharTS absorption sensitive to changes in protein conformations and protein-protein interactions. Recently, luminescence associated with these charge transfer transitions was established, opening further avenues for application of ProCharTS to track protein oligomer formation and protein-ligand interactions. The function of ProCharTS Proteins are the workhorses in a living cell. Traditionally, the only functional groups in the protein that could absorb light in the near-UV-VIS region (250 to 320 nm) were thought to be the aromatic moieties in the side chain of the amino acids Trp, Tyr, and Phe. Aside from the disulfide bonds that display weak absorption — and in the absence of metal ions, cofactors, or prosthetic groups — proteins were considered optically silent in the 320- to 800-nm region. This created the impression that proteins are insensitive and featureless in the near-UV-VIS electromagnetic band. Early work by the authors, however, showed this was not the case1. For the first time, it was shown that nonaromatic but charged headgroups in the side chains of Asp, Glu, Lys, His, Arg (Figure 1) — along with phosphorylated hydroxyl groups in Ser, Thr, and Tyr — could absorb light in the 250- to 800-nm region of the spectrum2,3. Figure 1. ProCharTS absorption from 250 to 800 nm in proteins. The absorption spectra for PEST wildtype (wt)* and α3C (devoid of aromatic amino acids) in the 250- to 800-nm range (a). The absorption spectra for human serum albumin and hen egg white lysozyme (containing aromatic amino acids) in the 320- to 800-nm range (b). *PEST wt contains one phenylalanine. Courtesy of Amrendra Kumar. How ProCharTS originates Two main types of charge transfer transitions are involved in ProCharTS2. Protein backbone-side chain transfer (PB-SCT). PB-SCT occurs due to photoinduced electron transfer between a protein backbone and the charged headgroup of amino acid. Typically, it may involve electron transfer from the polypeptide backbone, which is a rich source of electrons, to the positively charged primary amino group (−NH3+) (Figure 2a). Here, the backbone acts as electron donor, while the cationic Lys headgroup acts as the acceptor. Additionally, the donor can be the charged carboxylate anion (−COO−) of an Asp or Glu, while the acceptor is the polypeptide backbone. In both cases, the bonds between the donor and the acceptor act as a bridge to facilitate the electron transfer. Normally, the energy gap between the highest occupied molecular orbital (HOMO) of the donor and the lowest unoccupied molecular orbital (LUMO) of the acceptor is quite large for charged residue in side chain, curtailing absorption to shorter wavelengths. For example, this energy gap could be ~6.3 eV for a monomeric Lys-backbone pair (Figure 2a) or ~4.5 eV for a monomeric Glu-backbone pair. However, when a cluster of like-charged atoms is present in close vicinity (0.3 to 0.5 nm), enabled by a folded protein (Figure 2b), the HOMO-LUMO energy gap decreases, resulting in a shift of absorption to longer wavelengths. Here, the proximity between like-charged atoms destabilizes the ground state due to repulsion, but photoinduced electron transfer can stabilize the excited state by the gain (in the case of Ly-Lys) or loss (for Glu-Glu) of electrons from the charge cluster. However, if the charged atoms drift apart to longer distances (>0.8 nm), the energy gap increases. Thus, the intensity and position of the ProCharTS absorption band is sensitive to the changes in the distance between clusters of like-charged atoms. Side chain-side chain transfer (SC-SCT). In SC-SCT, the photoinduced electron transfer occurs from a negatively charged headgroup of amino acid (such as glutamate or aspartate) to a positively charged headgroup of amino acid (such as lysine) (Figure 2c). Thus Glu/Asp is the donor while Lys is the acceptor. Interestingly, the bridge in this case with oppositely charged donor-acceptor pairs is the intervening solvent space between the donor and acceptor. Figure 2. A schematic of photoinduced electron transfer in a monomeric folded polypeptide chain with a Lys monomer (a), a Lys-Lys dimer (b), and a Lys-Glu dimer (c). H: His; K: Lys; R: Arg; E: Glu; D: Asp. Courtesy of Amrendra Kumar. Remarkably, the HOMO-LUMO energy gap for SC-SCT is inversely proportional to the distance between the oppositely charged atoms, unlike with Figure 2b. For example, when the charged Lys amino and Glu carboxylate atoms are close enough (0.2 to 0.3 nm) to form a salt bridge (Figure 3c), the HOMO-LUMO gap is high, and absorption is in the far-UV (Figure 3d-1). However, when the oppositely charged atoms are slightly far apart (0.5 to 0.6 nm), as in Figure 3a, the energy gap between the HOMO of the donor and the LUMO of the acceptor is dramatically reduced. A range of distances between oppositely charged atoms in a folded protein can thus result in a display of a long tail in the absorption band until 800 nm (Figure 3d-3 and Figure 1a). Figure 3. The influence of distance between oppositely charged atoms of the Lys-Glu pair on the HOMO-LUMO (highest occupied molecular orbital-lowest unoccupied molecular orbital) energy gap. The HOMO-LUMO energy gaps for Lys-Glu pairs that are separated at different distances from each other (a, b, c). A schematic representing the dependence of ProCharTS wavelength upon the spatial distance between the interacting Lys and Glu charged atoms (d). DS: distally separated. Courtesy of Amrendra Kumar. Sensitivity to charge The presence of charge is essential in order for ProCharTS to be observed. If the charges in protein are altered by changing the pH of the aqueous medium, or if the charges are shielded by the added presence of salt ions, the ProCharTS absorption is affected4. To test the effect of salt and charge neutralization, the ProCharTS of α3C protein in NaCl was studied. The counter ions (Na+ or Cl−) were expected to screen the negative charge on the carboxylate anion in glutamate side chains, or the positive charge on lysine side chains, diminishing most of the Glu-Glu and Lys-Glu interactions. This explains the reduced intensity observed at longer wavelengths with increased salt (Figure 4). Figure 4. The effect of salt (NaCl) on ProCharTS. The absorption spectrum of α3C in the presence of various concentrations of NaCl in the 250- to 800-nm range. Courtesy of Anurag Priyadarshi. Sensitivity to structure Protein conformation. Earlier, it was explained that ProCharTS absorption is dependent on the distance between charged atoms in a cluster. This sensitivity to distance immediately enables researchers to detect changes, which are key to a whole range of biomedical processes, in protein conformation by simply measuring the protein absorption spectrum. The change in conformation of protein from natively folded to unfolded conformations could result in loss of proximity among charged atoms (and thus ionic bonding) in interacting side chains. Similarly, the transition from unfolded to folded conformation could increase such proximity. This change in the population of interacting side chains will be reflected as a change in ProCharTS absorption intensity, depending upon the decrease or increase in the population of interacting side chains. For example, intrinsically disordered proteins that are rich in charged residues generally acquire folded conformation in an acidic environment, where the hydrogen bonds are broken, and this collapsed structure can drive an increase in ProCharTS absorption. To understand this characteristic, the authors studied the intrinsically disordered PEST region (PEST wild-type, or wt) — a region rich in proline (P), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T) — from human c-Myc oncoprotein4. As expected, PEST wt — upon exposure to an acidic environment (pH 3) — displayed an increase in ProCharTS absorption due to its partial compaction and folding. The formation of partially folded conformation was corroborated by circular dichroism spectra, confirming that the change in ProCharTS was indeed due to a change in protein conformation (Figure 5). This simple experiment proved the sensitivity of ProCharTS toward change in protein conformation. Figure 5. ProCharTS is sensitive to changes in protein conformation. The pH-dependent change in the ProCharTS of PEST wt (150 μM) between 325 and 800 nm (a), and between 250 and 325 nm (inset). The break in the x-axis of the inset is between 294 and 295 nm. The pH-dependent change in the molar ellipticity of identical samples (b). Courtesy of Mohd. Ziauddin Ansari. Protein oligomeric state (aggregation). ProCharTS can also successfully track protein aggregation4. Protein aggregation is a protein-protein interaction in which possibilities of immense new intermolecular contacts arise. This aggregation has been identified as a potential cause of many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. The population of such new intermolecular contacts depends upon the type (for example, amorphous or fibrillar) and size (for example, oligomers or larger aggregates) of soluble protein aggregates. As a proof of concept, the authors’ group considered aggregating hen egg white lysozyme (HEWL) protein in both alkaline and acidic conditions. HEWL normally harbors ~21% of charged residues in its protein sequence and displays minimal ProCharTS (Figure 1b) in its native conformation. However, upon aggregation, the same protein displayed enhanced ProCharTS absorption, indicating an enormous increase in the population of interacting side chains within protein aggregates (Figure 6). This change in ProCharTS absorption was proved to be caused by protein aggregation, because the presence of protein aggregates was independently confirmed by a thioflavin T (ThT) assay. Further abolishing protein aggregation in HEWL by inhibiting disulfide bond formation also diminished increase in ProCharTS absorption intensity4. Figure 6. The effect of protein aggregation on ProCharTS. Time-dependent absorption spectra for hen egg white lysozyme (50 μM) aggregates formed at an acidic pH (pH 5; citrate buffer; 0.1 M). Courtesy of Amrendra Kumar. Surprisingly, preliminary lab data suggests that ProCharTS signals are more sensitive to early oligomers of protein, while ThT is generally believed to be more sensitive to the presence of mature fibrillar aggregates (amyloids). Hence, ProCharTS may have an advantage over other classical methods for detecting early oligomeric transitions in proteins as a label-free intrinsic probe. This needs further investigation. ProCharTS applications In a folded protein, charged atoms can be brought closer by the 3D fold of the protein. This process can trigger a significant increase in populations of charged atoms in close proximity to each other ( Protein post-translational modifications serve as flags or signals for regulating protein function inside the cell. However, detecting these modifications in a protein is not easy. Direct readouts are not possible without purifying the modified protein and performing mass spectrometry. Current approaches include the use of an (expensive) antibody designed to bind to the newly added chemical entity in the modified protein. But mass spectrometry facilities are expensive to use, and sample preparation steps for this approach are tedious to perform. Fortunately, many of the post-translational modifications involve modifying lysine residues such as acetylation or methylation. Acetylation of the amino group in lysine results in loss of charge, making it detectable using ProCharTS5. Interestingly, phosphorylation of otherwise uncharged residues — such as Ser, Thr, or Tyr (in protonated form) — can result in changed absorption arising from charged phosphoryl groups3. More laboratory studies are needed to validate that this approach is capable of detecting post-translational modifications. The binding of DNA or RNA with protein involves the interaction of phosphate anions in DNA/RNA with oppositely charged Arg or Lys residues in protein. Such interactions are often monitored indirectly by using techniques such as gel-shift assays. A direct spectroscopy-based readout using FRET (Förster resonance energy transfer) can be a tedious experiment, further complicated by unnatural covalent modifications in DNA and protein. ProCharTS could prove useful for monitoring these interactions. Since DNA and RNA binding can neutralize the charge in cationic residues in protein, the binding of nucleic acid can result in the decrease in ProCharTS absorption intensity at wavelengths longer than 300 nm (Figure 7b), where nucleic acid absorption is negligible. Thus, this can enable direct monitoring of protein-nucleic acid interactions. Such monitoring is at the heart of thriving research in chromatin biology, histone-DNA interactions, DNA repair, and replication. Figure 7. Schematics of potential applications of ProCharTS. The effect of ProCharTS on protein folding (a) and on DNA-protein interactions (b). Courtesy of Amrendra Kumar. Luminescence Weak intrinsic luminescence in the visible region, arising from charge recombination on excitation at protein charge transfer bands, has been observed6. It was shown that the luminescence intensity is primarily dependent on the molar extinction coefficient at the excitation wavelength employed. The quantum yields of ProCharTS luminescence for all charged proteins investigated so far have been low ( The emergence of intrinsic luminescence from ProCharTS bands can be utilized for the applications described above. However, considering the higher sensitivity of luminescence as compared to absorbance, more elaborate and detailed information could be made available. Currently, work is underway to determine how luminescence data can complement the absorption results. Label-free probe Labeling proteins with extrinsic probes to study their structure and dynamics is popular in life science research. However, the labeling process is tedious and causes structural perturbation to the natural state of the protein, which may reduce the reliability of results. Further, the synthesis of the labels is also a tedious process, and it involves the application of toxic reagents that can harm the environment. Thus, label-free probes such as ProCharTS promote green chemistry and sustainability. Importantly, ProCharTS permits investigations on protein structures in their natural state. Label-free techniques such as ProCharTS are ideally suited for applications that take place inside living cells for two reasons. First, it is extremely difficult to introduce labeled probes inside living cells. Such probes are currently either microinjected, with traumatic consequences to the cell, or as with green fluorescent protein, the probes are endogenously expressed in the cell. Second, ProCharTS displays absorption in the 600- to 800-nm region, where most other cellular biomolecules are optically silent. Because no label-free approaches are currently available for detecting protein aggregates in neurodegenerative diseases such as Alzheimer’s or Parkinson’s, it may be worthwhile to explore using ProCharTS for these applications. Meet the authors Amrendra Kumar, Ph.D., completed his doctorate in biosciences and bioengineering from the Indian Institute of Technology (IIT) Guwahati in India in 2020. His main research interest involves the biophysics and intrinsic luminescence of biological macromolecules; email: k.amrendra@alumni.iitg.ac.in. Rajaram Swaminathan, Ph.D., is a senior professor in the Department of Biosciences and Bioengineering at IIT Guwahati. His group employs spectroscopic techniques such as fluorescence and charge transfer spectra to investigate the structure, function, and dynamics of proteins; email: rsw@iitg.ac.in. References 1. L. Homchaudhuri and R. Swaminathan (2004). Near ultraviolet absorption arising from lysine residues in close proximity: a probe to monitor protein unfolding and aggregation in lysine-rich proteins. Bull Chem Soc Japan, Vol. 77, pp. 765-769. 2. S. Prasad et al. (2017). Near UV-visible electronic absorption originating from charged amino acids in a monomeric protein. Chem Sci, Vol. 8, pp. 5416-5433. 3. I. Mandal et al. (2018). Optical backbone-sidechain charge transfer transitions in proteins sensitive to secondary structure and modifications. Faraday Discuss, Vol. 207, pp. 115-135. 4. M.Z. Ansari et al. (2018). Protein charge transfer absorption spectra: an intrinsic probe to monitor structural and oligomeric transitions in proteins. Faraday Discuss, Vol. 207, pp. 91-113. 5. K.H. Jong et al. (2019). Low energy optical excitations as an indicator of structural changes initiated at the termini of amyloid proteins. Phys Chem Chem Phys, Vol. 21, pp. 23931-23942. 6. A. Kumar et al. (2020). Weak intrinsic luminescence in monomeric proteins arising from charge recombination. J Phys Chem B, Vol. 124, pp. 2731-2746.