Label-Free Tracking of Hepatitis B Virus Core Protein Capsid Assembly in Real-Time Using Protein Charge Transfer Spectra.

Hepatitis B virions are double-shelled particles, with a diameter of 40-42 nm, consisting of a nucleocapsid called the HBV core protein (HBV Cp). It is an ordered assembly of 90-120 homodimers arranged in an icosahedral symmetry. Both the full-length HBV Cp and the first-149 residue domain, HBV Cp149, can spontaneously assemble in vitro into capsids with 120 Cp dimers (T = 4) or 90 Cp dimers (T = 3), triggered by high ionic strength of 0.25-0.5 M NaCl. The assembly disassembly of HBV Cp149 capsids are generally studied by light scattering, size-exclusion chromatography, atomic force microscopy, transmission electron microscopy, and other high-end expensive techniques. Here, we report a simple, yet robust, label-free technique exploiting protein charge transfer spectra (ProCharTS) to monitor the capsid assembly in real-time. ProCharTS absorption in the near UV-visible region (250-800 nm) arises when photoinduced electron transfer occurs from HOMO of COO- in glutamate (donor) to LUMO of NH3+ in lysine or polypeptide backbone (acceptor) of the protein. Alternatively, it can also occur from polypeptide backbone (donor) to acceptor in arginine, histidine, or lysine cation. ProCharTS is observed profusely among proximal charge clusters in folded proteins. Here, we show that, ProCharTS absorption among growing HBV capsids is amplified when HBV Cp homodimers assemble, generating new contacts among charged residues in the dimer-dimer interface. We notice a time-dependent sigmoidal increase in ProCharTS absorbance and luminescence during capsid formation in comparison to pure dimers. Additionally, a combined approach of anisotropy-based fluorescence assay is reported, where an increased fluorescence anisotropy was observed in capsids as compared to native and unfolded dimers. We conclude that ProCharTS can serve as a sensitive label-free tool for rapid tracking of capsid assembly in real-time and characterize the assembled capsids from dimers.

Early events during the aggregation of Aß16-22-derived switch-peptides tracked using Protein Charge Transfer Spectra.

BACKGROUND: Understanding Aß aggregation and inhibiting it at early stages is of utmost importance in treating Alzheimer's and other related amyloidogenic diseases. However, majority of the techniques to study Aß aggregation mainly target the late stages; while those used to monitor early stages are either expensive, use extrinsic dyes, or do not provide information on molecular level interactions. Here, we investigate the early events of Aß16-22(KLVFFAE) aggregation using Aß16-22 derived switch-peptides (SwPs) through a novel label-free approach employing Protein Charge Transfer Spectra (ProCharTS). RESULTS: When pH is increased from 2 to 7.2, the Aß-derived switch peptides undergo controlled self-assembly, where the initial random coil peptides convert into ß-sheet. We leveraged the intrinsic absorbance/luminescence arising from ProCharTS among growing peptide oligomers to observe the aggregation kinetics in real-time. In comparison to monomer, the lysine and glutamate headgroups in the peptide oligomer are expected to come in proximity enhancing ProCharTS intensity due to photoinduced electron transfer. With a combination of Aß-derived switch-peptides and ProCharTS, we obtained structural insights on the early stages of Aß-derived SwP aggregation in four unique peptides. Increase in scatter corrected ProCharTS absorbance (250-500 nm) and luminescence (320-720 nm) along with decreased mean luminescence lifetime (2.3-0.8 ns) characterize the initial stages of aggregation monitored for 1-96 h depending on the peptide. We correlated the results with Circular Dichroism (CD), 8-anilino-1-naphthalenesulfonic acid (ANS) and Thioflavin T (ThT) measurements. SIGNIFICANCE: We demonstrate ProCharTS as an intrinsic analytical probe with following advantages over other conventional methods to track aggregation: it is a label-free probe; it's intensity can be measured using a UV-Vis spectrophotometer; it is more sensitive in detecting the early molecular events in aggregation compared to ANS and ThT; and it can provide information on specific contacts made between charged headgroups of Lysine/Glutamate in the oligomer.

Protein charge transfer spectra in a monomeric protein with no lysine.

UV-Visible absorption and luminescence originating from non-aromatic groups in proteins is being intensely investigated today. Earlier work has shown that non-aromatic charge clusters in a folded monomeric protein can collectively act like a chromophore. Incident light in the near UV-Visible wavelength causes photoinduced electron transfer from the Highest Occupied Molecular Orbital (HOMO) of the electron-rich donor (like a carboxylate anion) to the Lowest Unoccupied Molecular Orbital (LUMO) of the electron-deficient acceptor (like a protonated amine or the polypeptide backbone) in the protein giving rise to absorption spectra in the 250-800 nm range referred to as Protein Charge Transfer Spectra (ProCharTS). The transferred electron can relax back from the LUMO to fill up the hole in the HOMO by a charge recombination process, emitting weak ProCharTS luminescence. Previous studies in monomeric proteins displaying ProCharTS absorption/luminescence always involved lysine-containing proteins. The lysine (Lys) sidechain plays a dominant role in ProCharTS; however, experimental evidence for ProCharTS among proteins/peptides devoid of Lys is lacking. Recently, the absorption features of charged amino acids have been examined using time-dependent density functional theory calculations. In this study, we show that amino acids: arginine (Arg), histidine (His) and aspartate (Asp); homo-polypeptides: poly-arginine and poly-aspartate; and a protein: Symfoil PV2 that is rich in Asp, His and Arg, but lacks Lys, profusely display ProCharTS. The folded Symfoil PV2 protein displayed maximum ProCharTS absorptivity in the near UV-Vis region, in comparison to the homo-polypeptides and amino acids. Furthermore, features like overlapping ProCharTS absorption spectra, decreasing ProCharTS luminescence intensity with longer excitation wavelength, large Stokes shift, multiple excitation bands and multiple luminescence lifetime components appeared to be conserved across peptides, proteins and amino acids studied. Our results underscore the utility of ProCharTS as an intrinsic spectral probe to monitor the structure of any protein that is rich in charged amino acids.

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.