Disruption of Spatial Proximities among Charged Groups in Equilibrium-Denatured States of Proteins Tracked Using Protein Charge Transfer Spectra.

The absorption and luminescence originating from protein charge transfer spectra (ProCharTS) depend on the proximity between multiple charged groups in a protein. This makes ProCharTS absorbance/luminescence intensity a sensitive probe for detecting changes in the protein structure, which alter the proximity among charged groups in the protein. In this work, ProCharTS absorbance of charge-rich proteins like human serum albumin (HSA), α3C, and α3W was used to monitor structural changes upon chemical denaturant-induced protein unfolding under equilibrium conditions. The denaturation midpoints were estimated using nonlinear regression analysis. For HSA, absorbance at 325 and 340 nm estimated the GdnHCl-induced denaturation midpoints to be 0.80 and 0.61 M, respectively. A similar analysis of α3C and α3W ProCharTS absorbance yielded denaturation midpoints of 0.88 and 0.86 M at 325 nm and 0.96 and 0.66 M at 340 nm, respectively. A previously reported molten globule-like state in the GdnHCl-induced HSA unfolding pathway was detected by the increase in HSA ProCharTS absorbance at 0.5 M GdnHCl. To validate the above results, protein unfolding was additionally monitored using conventional methods like circular dichroism (CD), Trp, and dansyl fluorescence. Our results suggest that disruption of charged amino acid sidechain contacts as revealed by ProCharTS occurs at lower denaturant concentrations compared to the loss of secondary/folded structure monitored by CD and fluorescence. Further, HSA ProCharTS absorbance at 315-340 nm revealed that tertiary contacts among charged residues were disrupted at lower GdnHCl concentrations compared to sequence adjacent contacts. Our data underscore the utility of ProCharTS as a novel label-free tool to track unfolding in charge-rich proteins.

Cold atmospheric plasma driven self-assembly in serum proteins: insights into the protein aggregation to biomaterials.

The self-assembly of proteins is crucial in many biomedical applications. This work deals with understanding the role of cold atmospheric plasma (CAP) on the self-assembly of two different proteins present in the serum - BSA and hemoglobin and to elucidate the process associated with the direct application of physical plasma on or in the human (or animal) body, which has implications in therapeutics. The work has been corroborated by several spectroscopic studies such as fluorescence spectroscopy, circular dichroism spectroscopy, and SEM analysis. Through steady-state fluorescence spectroscopy and by following the tryptophan fluorescence, we observed that the emission intensity was quenched for the protein when treated with plasma radiation. Circular dichroism spectroscopy revealed that the structure of the protein was altered both in the case of BSA and hemoglobin. N-Acetyl tryptophanamide (NATA), which resembles the tryptophan in the protein, was treated with CAP and we observed the similar quenching of fluorescence as in the proteins, indicating that the protein underwent self-assembly. Time-resolved fluorescence spectroscopy with a decrease in the lifetime revealed that the protein self-assembly was promoted with CAP treatment, which was also substantiated by SEM micrographs. The ROS/RNS produced in the CAP has been correlated with the protein self-assembly. This work will help to design protein self-assembled systems, and in the future, may bring possibilities of creating novel biomaterials with the help of plasma radiation.

Role of Charged Amino Acids in Sullying the Fluorescence of Tryptophan or Conjugated Dansyl Probe in Monomeric Proteins.

When Trp/dansyl probe conjugated to a monomeric protein is photoexcited, it is assumed that all emitted fluorescence originates solely from them. In this work, we show that hidden unconventional intrinsic chromophores (called ProCharTS) that originate from confined charge clusters in the protein can contaminate Trp/dansyl emission. Previous work has shown that charge recombination among charge-separated excited states of monomeric proteins, rich in charged residues, can emit weak luminescence (300-700 nm) overlapping with ProCharTS absorption (250-800 nm) and Trp (300-400 nm) and dansyl (400-600 nm) emission. We examine how this overlap taints the fluorescence arising from Trp/dansyl. We compared the effect of dense aqueous solutions of amino acids, Lys/Glu/Asp/Arg/His, on the fluorescence intensity decay/spectrum of N-acetyl-l-tryptophan amide (NATA). Significant broadening on the red side of Trp emission spectrum was observed solely in the presence of lysine, which appeared to be the most potent in altering the mono-exponential fluorescence decay of NATA. Interestingly, NATA in the presence of proteins α3C and dehydrin (DHN1), which are rich in Lys residues, showed substantial deviation from mono-exponential fluorescence decay in contrast to PEST wt and Symfoil-4P pv2, which lack Lys residues. Remarkably, Trp emission spectra among charge-rich proteins like α3W, PEST M1, and DHN1 CW1 were altered on the red side of Trp emission. Emission spectrum of dansyl-labeled human serum albumin (HuSA) was broadened and its fluorescence quenched with gradual addition of excess unlabeled HuSA, which displays bountiful ProCharTS luminescence. Our results unveil the additive influence of ProCharTS luminescence on Trp/dansyl emission with no measurable evidence of energy transfer.

Weak Intrinsic Luminescence in Monomeric Proteins Arising from Charge Recombination.

We had earlier reported on the presence of broad UV-vis electronic absorption (250-800 nm) in a monomeric protein rich in charged but lacking aromatic amino acids, referred to as Protein Charge Transfer Spectra (ProCharTS). Specifically, it was shown that the cationic amino/anionic carboxylate head groups of Lys/Glu side chains act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other. In this work, we show that such excitations produce weak intrinsic luminescence in proteins originating from charge recombination. We investigated aqueous solutions of proteins with varying abundance of charged amino acids, like human serum albumin (HuSA) and hen lysozyme, and intrinsically disordered proteins, like PEST fragment of human c-Myc protein, α-synuclein, and dehydrin. The absorbance and luminescence in all protein samples were a linear function of the concentration (0-50 µM) employed, confirming their origin from a monomeric species. The slope of the luminescence/[protein] plot directly correlated with the fraction of charged amino acids present in protein. Specifically, the higher slope in proteins like HuSA was chiefly accounted by a large molar extinction coefficient rather than quantum yield. This coefficient directly correlates with the population of charged side-chain head groups lying in close spatial proximity in the protein, contributed by the three-dimensional (3D) fold of the polypeptide. ProCharTS luminescence parameters appear conserved across proteins. These include overlapping excitation/emission spectra, large Stokes shifts (14 000-3000 cm-1) that decrease with increasing excitation wavelength, low quantum yields (0.002-0.026) indicating poor radiative recombination efficiency, and multiexponential decays (mean lifetimes = 0.4-2.9 ns).

Protein charge transfer absorption spectra: an intrinsic probe to monitor structural and oligomeric transitions in proteins.

Protein Charge Transfer Spectra (ProCharTS) originate 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. The absorption band intensities in ProCharTS at wavelengths of 250-800 nm are dependent on the 3D spatial proximity of these charged functional groups across the protein. Intrinsically disordered proteins (IDPs) are an important class of proteins involved in signalling and regulatory functions in the eukaryotic cell. IDPs are rich in charged amino acids, but lack structure-promoting intrinsic spectral probes like Tyr or Trp in their sequences, making their structural characterisation difficult. Here, we exploit the richness of charged amino acid populations among IDPs (like the PEST fragment of human c-Myc, its mutant and dehydrin from maize) to sense structural transitions in IDPs using ProCharTS absorption spectra. Conformational changes induced in the protein by altering the pH and temperature of the aqueous medium were monitored by ProCharTS and confirmed by CD spectra. Further, the utility of ProCharTS to detect protein aggregation was examined using Hen Egg-White Lysozyme (HEWL) protein. The results revealed that in the presence of Trp/Tyr, ProCharTS absorbance was substantially reduced, specifically at wavelengths where the absorption by Trp or Tyr was near its maximum. Significant changes in the ProCharTS spectra were observed with changing pH in the range of 3-11, which correlated with changes in the secondary structure of the PEST fragment. Importantly, the absorbance at 280 nm, which is often employed as a measure of protein concentration, was profoundly altered by changes in ProCharTS intensity in response to changing the pH in dehydrin. The ProCharTS intensity was sensitive to temperature-induced changes in the secondary structures of the PEST fragments between 25-85 °C. The presence of 0.25 M NaCl or KCl in the medium also altered the ProCharTS spectrum. Finally, an increase in ProCharTS absorbance with time in HEWL at pH 2 directly correlated with the growth of HEWL aggregates and amyloid fibrils, as confirmed by the increasing thioflavin T fluorescence. Taken together, our work highlights the utility of ProCharTS as a label-free intrinsic probe to monitor changes in protein charge, structure and oligomeric state.