The effect of chrysin binding on the conformational dynamics and unfolding pathway of human serum albumin.

Studies on the interactions between ligands and proteins provide insights into how a possible medication alters the structures and activities of the target or carrier proteins. The natural flavonoid aglycone Chrysin (CHR) has demonstrated anti-inflammatory, antioxidant, antiapoptotic, neuroprotective, and antineoplastic effects, both in vitro and in vivo. In this work, we investigated the impact of CHR binding on the as-yet-unexplored conformation, dynamics, and unfolding mechanism of human serum albumin (HSA). We determined CHR binding to HSA domain-II with the association constant (Ka) of 2.70 ± 0.21 × 105 M-1. The urea-induced sequential unfolding mechanism of HSA was used to elucidate the debatable binding location of CHR. CHR binding induced both secondary and tertiary structural alterations in the protein as studied by far-UV circular dichroism and intrinsic fluorescence spectroscopy. Red edge excitation shift (REES) indicated a decrease in conformational dynamics of the protein on the complex formation. This suggested an ordered compact and spatial arrangement of the CHR-boundmolecule. The binding of CHR was found to significantly modulate the urea-induced unfolding pathway of HSA. Urea-induced unfolding pathway of HSA became a two-state process (N-U) from a three-state process (N-I-U). The interaction of CHR is found to increase the thermal stability of the protein by ∼4 °C. This study focuses on the fundamental sciences and demonstrates how prospective medication compounds can alter the dynamics and stability of protein structure.

In-depth investigation of the effect of pH on the autofluorescence properties of DPF3b and DPF3a amyloid fibrils.

Double PHD fingers 3 (DPF3) protein exists as two splicing variants, DPF3b and DPF3a, the involvement of which in human cancer and neurodegeneration is beginning to be increasingly recognised. Both isoforms have recently been identified as intrinsically disordered proteins able to undergo amyloid fibrillation. Upon their aggregation, DPF3 proteins exhibit an intrinsic fluorescence in the visible range, referred to as deep-blue autofluorescence (dbAF). Comprehension of such phenomenon remaining elusive, we investigated in the present study the influence of pH on the optical properties of DPF3b and DPF3a fibrils. By varying the excitation wavelength and the pH condition, the two isoforms were revealed to display several autofluorescence modes that were defined as violet, deep-blue, and blue-green according to their emission range. Complementarily, analysis of excitation spectra and red edge shift plots allowed to better decipher their photoselection mechanism and to highlight isoform-specific excitation-emission features. Furthermore, the observed violation to Kasha-Vavilov's rule was attributed to red edge excitation shift effects, which were impacted by pH-mediated H-bond disruption, leading to changes in intramolecular charge and proton transfer, or π-electrons delocalisation. Finally, emergence of different autofluorescence emitters was likely related to structurally distinct fibrillar assemblies between isoforms, as well as to discrepancies in the amino acid composition of their aggregation prone regions.

Red edge excitation shift spectroscopy is highly sensitive to tryptophan composition.

Red edge excitation shift (REES) spectroscopy relies on the unique emission profiles of fluorophore-solvent interactions to profile protein molecular dynamics. Recently, we reported the use of REES to compare the stability of 32 polymorphic IgG antibodies natively containing tryptophan reporter fluorophores. Here, we expand on this work to investigate the sensitivity of REES to variations in tryptophan content using a subset of IgG3 antibodies containing arginine to tryptophan polymorphisms. Structural analysis revealed that the additional tryptophan residues were situated in highly solvated environments. Subsequently, REES showed clear differences in fluorescence emission profiles when compared with the unmutated variants, thereby limiting direct comparison of their structural dynamics. These findings highlight the exquisite sensitivity of REES to minor variations in protein structure and tryptophan composition.

Multi-action of a Fluorophore in the Sight of Light: Release of NO, Emergence of FONs, and Organelle Switching.

Light, as an external stimulus, has begun to engage a phenomenal role in the diverse field of science. Encouraged by recent progress from biology to materials chemistry, various light-responsive fluorescent probes have been developed. Herein, we present a 1,8-naphthalimide-based probe NIT-NO2 capable of releasing nitric oxide (NO) along with the formation of fluorescent organic nanoparticles (FONs) upon exposure to near-visible UV light. By synthesizing the photoproduct NIT-OH, we unveiled that initially NIT-NO2 released NO and converted to NIT-OH, while prolonged irradiation led to the formation of FONs that is corroborated by the red-edge excitation shift as well as microscopic investigation. Finally, we have successfully applied NIT-NO2 and NIT-OH for specific labeling of lipid droplets and plasma membranes, respectively, and demonstrated the switching from lipid droplets to plasma membranes by using light as a stimulus. These two probes show unique imaging applications inside the cells depending on the polarity and hydrophobicity of the environment. This work paves a fascinating way for the generation of excitation-dependent FONs from a small organic fluorophore and highlights its potency as an exclusive imaging tool.

A Thermodynamic Model for Interpreting Tryptophan Excitation-Energy-Dependent Fluorescence Spectra Provides Insight Into Protein Conformational Sampling and Stability.

It is now over 30 years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. While there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the tryptophan REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides, to multitryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibodies in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating biomolecular simulation with experimental data to yield novel insights.

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein-Ligand Complexes.

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.

Novel insights in linking solvent relaxation dynamics and protein conformations utilizing red edge excitation shift approach.

Protein hydration dynamics plays an important role in many physiological processes since protein fluctuations, slow solvation, and the dynamics of hydrating water are all intrinsically related. Red edge excitation shift (REES) is a unique and powerful wavelength-selective (i.e. excitation-energy dependent) fluorescence approach that can be used to directly monitor the environment-induced restriction and dynamics around a polar fluorophore in a complex biological system. This review is mainly focused on recent applications of REES and a novel analysis of REES data to monitor the structural dynamics, functionally relevant conformational transitions and to unmask the structural ensembles in proteins. In addition, the novel utility of REES in imaging protein aggregates in a cellular context is discussed. We believe that the enormous potential of REES approach showcased in this review will engage more researchers, particularly from life sciences.

Effect of pH and denaturants on the fold and metal status of anthrax lethal factor.

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10-11 mM of the surfactant n-tetradecyl-ß-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3-18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4-5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime.

Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma.

Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigated in the native state, in the presence of PrC and phosphatidylcholines (PCs) with short (valeryl, C-5) and long (myristoyl, C-14) chains, and upon denaturation using fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) measurements. The results obtained show that the environment of tryptophan residues in HSP-1/2 is more heterogeneous as compared to that in PDC-109. Binding of choline containing ligands afforded a protection to the tryptophan residues with the shielding order being: PrC≤divalaroyl PC

The red edge excitation shift phenomenon can be used to unmask protein structural ensembles: implications for NEMO-ubiquitin interactions.

To understand complex molecular interactions, it is necessary to account for molecular flexibility and the available equilibrium of conformational states. Only a small number of experimental approaches can access such information. Potentially steady-state red edge excitation shift (REES) spectroscopy can act as a qualitative metric of changes to the protein free energy landscape (FEL) and the equilibrium of conformational states. First, we validate this hypothesis using a single Trp-containing protein, NF-κB essential modulator (NEMO). We provide detailed evidence from chemical denaturation studies, macromolecular crowding studies, and the first report of the pressure dependence of the REES effect. Combination of these data demonstrate that the REES effect can report on the 'ruggedness' of the FEL and we present a phenomenological model, based on realistic physical interpretations, for fitting steady-state REES data to allow quantification of this aspect of the REES effect. We test the conceptual framework we have developed by correlating findings from NEMO ligand-binding studies with the REES data in a range of NEMO-ligand binary complexes. Our findings shed light on the nature of the interaction between NEMO and poly-ubiquitin, suggesting that NEMO is differentially regulated by poly-ubiquitin chain length and that this regulation occurs via a modulation of the available equilibrium of conformational states, rather than gross structural change. This study therefore demonstrates the potential of REES as a powerful tool for tackling contemporary issues in structural biology and biophysics and elucidates novel information on the structure-function relationship of NEMO and key interaction partners.

Importance of indole N-H hydrogen bonding in the organization and dynamics of gramicidin channels.

The linear ion channel peptide gramicidin represents an excellent model for exploring the principles underlying membrane protein structure and function, especially with respect to tryptophan residues. The tryptophan residues in gramicidin channels are crucial for the structure and function of the channel. In order to test the importance of indole hydrogen bonding for the biophysical properties of gramicidin channels, we monitored the effect of N-methylation of gramicidin tryptophans, using a combination of steady state and time-resolved fluorescence approaches along with circular dichroism spectroscopy. We show here that in the absence of the hydrogen bonding ability of tryptophans, tetramethyltryptophan gramicidin (TM-gramicidin) is unable to maintain the single stranded, head-to-head dimeric channel conformation in membranes. Our results show that TM-gramicidin displays a red-shifted fluorescence emission maximum, lower red edge excitation shift (REES), and higher fluorescence intensity and lifetime, consistent with its nonchannel conformation. This is in agreement with the measured location (average depth) of the 1-methyltryptophans in TM-gramicidin using the parallax method. These results bring out the usefulness of 1-methyltryptophan as a fluorescent tool to examine the hydrogen bonding ability of tryptophans in proteins and peptides. We conclude that changes in the hydrogen bonding ability of tryptophans, along with coupled changes in peptide backbone structure induce the loss of single stranded ß(6.3) helical dimer conformation. These results agree with earlier results from size-exclusion chromatography and single-channel measurements for TM-gramicidin, and confirm the importance of indole hydrogen bonding for the conformation and function of ion channels and membrane proteins.

Interaction of bee venom toxin melittin with ganglioside GM1 bicelle.

Melittin is a bee venom toxin that can act as antimicrobial peptide. Gangliosides are glycosphingolipids that help maintain membrane structure and organization as well as act as anchors for lectins, toxins, pathogens and antimicrobial peptides. Here we investigate interaction of melittin with fast tumbling isotropic control DMPC/CHAPS bicelles and ganglioside doped DMPC/CHAPS/GM1 bicelles. DOSY result shows that larger percentage of peptide binds to GM1 containing bicelles than that of the control PC bicelles. Bound peptide induces leakage of the bicelles entrapped carboxyfluorescein. Percentage of leakage is higher from control PC bicelles than that of the GM1 containing bicelles. In the presence of control PC bicelles melittin acquired fully α-helical structure. But in the presence of GM1 containing bicelles the peptide is not fully α-helical i.e., some random coil structure is present in this folded form. The present study shows that GM1 has an effect on membrane active antimicrobial peptide melittin.