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A schematic representation of chrysin (CHR) binding induced conformational dynamics and stability moderation in human serum albumin (HSA). [Display omitted] • The ligand binding-induced conformational alterations of protein are primary in drug discovery methods. • This study aims to elucidate chrysin binding-induced conformational dynamics and stability of human serum albumin. • We report noteworthy changes in conformational dynamics of the protein on chrysin binding. • The chrysin binding increase the thermal stability of HSA and modulate the unfolding pathway. 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 (K a) 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-bound molecule. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Autofluorescence (AF) properties of DPF3 amyloid fibrils are sensitive to pH. • Fibrils display different AF modes defined as violet, deep-blue, and blue-green. • Red edge shift behaviours allowed to decipher isoform-specific optical features. • Diversity in AF emitters and photoselection likely relates to fibrillar morphology. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] Many current methods for detecting molecular-level heterogeneity are complex and require stringent data analysis, limiting their widespread use. Herein, we propose a novel edge effect, i.e., the shift of excitation spectra at the blue edge of emission (which we termed as Blue Edge Emission Shift, BEEmS), to perceive the structural heterogeneity. This method is simple and can be easily implemented with commonly available fluorimeter. Red edge excitation shift (REES), a related technique, is already in use, but like most other known techniques, its usefulness is constrained by its dependence on environmental rigidity. Our method does not suffer from this drawback significantly. We showed the generality of the proposed method taking various chemically and biologically heterogenous systems including molecular liquid, deep eutectic solvents, organic cavitand, micelle and protein. BEEmS certainly comes out as a more effective sensor of heterogeneity than REES in certain cases (like denatured protein, hydrophobic deep eutectic solvent and SDS micelle) where solvation time is not sufficiently slow to be detected by REES, but can be measured though BEEmS. Furthermore, unlike most existing techniques, domain-specific heterogeneity of a model multi-domain protein is successfully measured. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 < dimyristoyl PC. REES value obtained for HSP-1/2 (3.5 nm) is smaller than that of observed for PDC-109 (4 nm) and binding to PrC and DVPC reduced the REES to 1 nm. HSP-1/2 exhibits only partial unfolding with chemical denaturants with no cooperativity, whereas complete unfolding was observed in the presence of 10 mM dithiothreitol, indicating that disulfide linkages prevent complete unfolding of the protein. In the presence of PrC the transition midpoints shifted to higher concentrations of the denaturant together with a broadening of the sigmoidal transitions, indicating that ligand binding as well as polydispersity modulate the unfolding process. [ABSTRACT FROM AUTHOR]

The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general. [ABSTRACT FROM AUTHOR]

Abstract: The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling. [Copyright &y& Elsevier]

Abstract: Bovine α-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca2+ depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general. [Copyright &y& Elsevier]

Abstract: The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red. [Copyright &y& Elsevier]

Abstract: Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides. [Copyright &y& Elsevier]

Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes. [ABSTRACT FROM AUTHOR]

Dendrimers are a new class of polymeric materials. They are globular, highly branched, monodisperse macromolecules. Due to their structure, dendrimers promise to be new, effective biomedical materials as oligonucleotide transfection agents and drug carriers. More information about biological properties of dendrimers is crucial for further investigation of dendrimers in therapeutic applications.In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine serum albumin (BSA) was examined. PAMAM dendrimers are based on an ethylenediamine core and branched units are constructed from both methyl acrylate and ethylenediamine. We used three types of PAMAM dendrimers with different surface groups (–COOH, –NH2, –OH). As BSA contains two tryptophan residues we were able to evaluate dendrimers influence on protein molecular conformation by measuring the changes in the fluorescence of BSA in the presence of dendrimers. Additionally experiments with a fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS) were carried out. The differential scanning calorimetry (DSC) was chosen to investigate impact on protein thermal stability upon the dendrimers.Our experiments showed that the extent of the interactions between BSA and dendrimers strongly depends on their surface groups and is the biggest for amino-terminated dendrimers. [Copyright &y& Elsevier]

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise ‘optically silent’ water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid–protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed. [Copyright &y& Elsevier]

The non-natural amino acids 7-azatryptophan (7AT) and 5-hydroxytryptophan (5HT) have come into significant recent prominence as novel intrinsic luminescence probes for protein structure, function and dynamics. Here, we examine the low temperature luminescence behaviours of these molecules and their respective chromophoric moieties 7-azaindole (7AI) and 5-hydroxyindole (5HI) in representative solvent media. To ascertain, in particular, the potential usefulness of 7AT and 5HT as phosphorescence probes for exploring protein environments with different hydrogen bonding characteristics, a comparison is made of the phosphorescence properties of 7AI and 5HI chromophores in frozen solutions of ethanol and ethyl acetate at 77 K. These solvent media have been chosen as representative models for polar protic and aprotic environments in proteins, respectively. Our findings indicate that one or more of the phosphorescence emission parameters (phosphorescence emission maxima, relative yield and phosphorescence lifetime) of 7AI and 5HI chromophores can serve as sensitive and discriminating probes of hydrogen bonding and related aspects of their surrounding environments. Furthermore, in a model viscous environment (glycerol at low temperatures) significant temperature dependence and red edge excitation shift (REES) effects are observed for the fluorescence emission of 7AT and its chromophoric moiety 7AI. This is consistent with pronounced dipolar relaxation properties of these molecules, and suggests interesting possibilities for exploiting REES in exploring their environmental rigidity in motionally constrained situations. [ABSTRACT FROM AUTHOR]