Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8-anilinonaphthalene-1-sulfonic acid (ANS) for local conformational analysis at the binding site in a crowding environment. Bovine serum albumin (BSA) concentration-dependent measurements were performed to assess the feasibility of ANS-ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS-ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature-dependent measurements were performed in dilute and crowding environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS-ICD spectral changes revealed lower temperature pre-denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self-crowding repulsive interactions. The established ANS-ICD method can provide valuable conformational insights into protein-ligand interactions in crowded cellular environments.

Dispersion Effect of Molecular Crowding on Ligand-Protein Surface Binding Sites of Escherichia coli RNase HI.

The molecular crowding effect on ligand-protein interactions, which plays several crucial roles in life processes, has been investigated using various models by adding crowding agents to mimic the intracellular environment. Several studies evaluating this effect have focused on the ligand-protein binding reaction of well-structured binding sites with rigid conformations. However, the crowding effect on flexible binding sites is not well-understood, especially in terms of the conformations. In this work, to elucidate the detailed molecular mechanism underlying the ligand-protein interactions with flexible binding sites on a protein surface, we studied the interaction between the basic protrusion of Escherichia coli ribonuclease HI (RNase HI) and 8-anilinonaphthalene-1-sulfonic acid (ANS). The RNase HI concentration-dependent measurement of ANS fluorescence combined with the multivariate analysis and the fluorescence vibronic structure analysis revealed an increase in the heterogeneous species with an increase in the protein concentration, which is a different behavior from that of proteins with rigid binding sites. This result indicates that ANS molecules bind to the additional binding sites because of the destabilization of the main sites by the excluded volume effect in a crowded environment. The fluorescence vibronic structure analysis yields a detailed molecular picture, indicating that the main species of ANS can have a distorted structure. On the other hand, some ANS molecules move to the minor binding sites of a different microenvironment to secure a stabilized structure. These spectroscopic analyses may show a hypothesis, suggesting that the decrease in the ΔG difference between the main and minor sites due to destabilization of the main binding site could lower the potential barrier between them, inducing the dispersion of binding pathways.

Revisiting the Rate-Limiting Step of the ANS-Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS-protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS-protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III.

The effect of salt on the electrostatic interaction of a protein is an important issue, because addition of salt affects protein stability and association/aggregation. Although adding salt is a generally recognized strategy to improve protein stability, this improvement does not necessarily occur. The lack of an effect upon the addition of salt was previously confirmed for the tenth fibronectin type III domain from human fibronectin (FN3) by thermal stability analysis. However, the detailed molecular mechanism is unknown. In the present study, by employing the negatively charged carboxyl triad on the surface of FN3 as a case study, the molecular mechanism of the inefficient NaCl effect on protein stability was experimentally addressed using spectroscopic methods. Complementary analysis using Raman spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence revealed the three-phase behavior of the salt-protein interaction between NaCl and FN3 over a wide salt concentration range from 100 mM to 4.0 M, suggesting that the Na+-specific binding to the negatively charged carboxyl triad causes a local conformational change around the binding site with an accompanying structural change in the overall protein, which contributes to the protein's structural destabilization. This spectroscopic evidence clarifies the molecular understanding of the inefficiency of salt to improve protein stability. The findings will inform the optimization of formulation conditions.

Spectroscopic Analysis of Protein-Crowded Environments Using the Charge-Transfer Fluorescence Probe 8-Anilino-1-Naphthalenesulfonic Acid.

The molecular behaviors of proteins under crowding conditions are crucial for understanding the protein actions in intracellular environments. Under a crowded environment, the distance between protein molecules is almost the same size as the molecular level, thus, both the excluded volume effect and short ranged soft chemical interaction on protein surface could induce the complicated influence on the protein behavior cooperatively. Recently, various kinds of analytical approaches from macroscopic to microscopic aspects have been made to evaluate the crowding effect. The method, however, has not been established to evaluate the surface specific interactions on protein surface. In this study, the analytical method to evaluate the crowding effect has been suggested by using a charge-transfer fluorescence probe, ANS. By employing the unique property of ANS attaching to charged residues on the surface of lysozyme, the crowding effect was focused, while the case was compared as a reference, in which ANS is confined in hydrophobic pockets of BSA. Consequently, the surface specific changes of fluorescence spectra were readily observed under the crowded environment, whereas the fluorescence spectra of ANS in protein inside did not change. This result suggests the fluorescence spectra of ANS binding to protein surface have the capability to estimate the crowding effect of proteins.

Ultrafast excited state dynamics of nonfluorescent cyclopheophorbide-a enol, a catabolite of chlorophyll-a detoxified in algae-feeding aquatic microbes.

For photosynthetic organisms that nourish the earth's biosphere, chlorophylls (Chls) are the major pigments utilized for light harvesting and primary charge separation. Although Chl molecules are effective photosensitizers, they are inevitably phototoxic to living organisms due to the facile generation of highly oxidative singlet oxygen (1O2) through triplet energy transfer from their photoexcited states to oxygen molecules. Such phototoxicity of Chls is a major problem for translucent microbes that feed on photosynthetic algae. Recently, it has been reported that the metabolic conversion of Chls-a/b to 132,173-cyclopheophorbide-a/b enols (cPPB-a/bEs) is the detoxification mechanism for algivorous protists. cPPB-a/bEs are colored π-conjugated cyclic tetrapyrroles but are nonfluorescent due to efficient nonradiative decay. In this study, femtosecond time-resolved transient absorption spectroscopy was applied to cPPB-aE with the aim of understanding its quenching mechanism. As a result, we have captured the ultrafast generation of an intermediate state (∼140 fs) that leads to the rapid internal conversion to the ground state (∼450 fs).

Behavior of Bovine Serum Albumin Molecules in Molecular Crowding Environments Investigated by Raman Spectroscopy.

The behavior of proteins in crowded environments is dominated by protein-crowder interactions (the entropic/excluded volume effect) and protein-protein interactions (the soft chemical effect). The details of these interactions, however, are not fully understood. In this study, the behavior of bovine serum albumin (BSA) in crowded environments, including high protein concentrations and in the presence of another protein, was investigated by Raman spectroscopy. A detailed analysis of the water, Tyr, and Phe Raman bands revealed that the excluded volume effect with an increase in the protein concentration changed the local environment of hydrophobic residues. In contrast, no specific changes to the secondary structure were observed from the analysis of the concentration dependence of the amide I band. BSA was experimentally shown to adopt a more compact state in the presence of the crowding agent. Moreover, H-D exchange experiments of the amide I band revealed that the intramolecular hydrogen bonds of BSA were strengthened in the presence of the protein crowder. Thus, the Raman spectroscopy results have revealed the molecular behavior of proteins in crowded environments by extracting information about the excluded volume effect, soft chemical interactions, and the hydration effect.

Assessment of the Protein-Protein Interactions in a Highly Concentrated Antibody Solution by Using Raman Spectroscopy.

PURPOSE: To investigate the protein-protein interactions of a highly concentrated antibody solution that could cause oligomerization or aggregation and to develop a better understanding of the optimization of drug formulations. METHODS: In this study, we used Raman spectroscopy to investigate the structure and interactions of a highly concentrated antibody solution over a wide range of concentrations (10-200 mg/mL) with the aid of a multivariate analysis. RESULTS: Our analysis of the amide I band, I 856 /I 830 of Tyr, and the relative intensity at 1004 cm(-1) of the Phe and OH stretching region at around 3000 cm(-1) showed that across this wide range of concentrations, the secondary structure of the IgG molecules did not change; however, short-range attractive interactions around the Tyr and Phe residues occurred as the distance between the IgG molecules decreased with increasing concentration. Analysis of the OH stretching region at around 3000 cm(-1) showed that these short-range attractive interactions correlated with the amount of hydrated water around the IgG molecules. CONCLUSIONS: Our data show that Raman spectroscopy can provide valuable information of the protein-protein interactions based on conformational approaches to support conventional colloidal approaches, especially for analyses of highly concentrated solutions.

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy.

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5-300 mg ml(-1)). No changes in the amide-I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm(-1) and the ratios of the intensities of the Tyr bands at 856 and 837 cm(-1), the Trp bands at 870 and 877 cm(-1), and the bands at 2940 (CH stretching) and 3420 cm(-1) (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high-concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals.

Investigation of the structure of water at hydrophobic and hydrophilic interfaces by angle-resolved TIR Raman spectroscopy.

To analyse the surface- or interface-specific molecular structure of a condensed molecular system, it is important to measure the spectra of molecules near the surface. Total internal reflection (TIR) Raman spectroscopy is a sensitive technique for surface or interfacial analysis because it retrieves spectra in the region within ca. 100 nm of a surface. However, since the width of the interface itself is often on a molecular scale (one to a few nm), conventional TIR Raman spectroscopy intrinsically lacks surface sensitivity. To overcome this problem, the combination of multiple-angle TIR Raman spectroscopy and principal component analysis (PCA) is expected to enable effective differentiation between the spectra of minute chemical species at the interface and those of dominant species. In the present study, angle-resolved TIR Raman spectroscopy with PCA was applied to SiO2/water and SAM/water interfaces to detect minute species located within a few nm of each interface. This method will likely lead to progress in various surface and interfacial analyses, not only those related to the structure of water, but also those used to determine the interactions among absorbed species.

Ultrafast Dynamics of a Solvatochromic Dye, Phenol Blue: Tautomerization and Coherent Wavepacket Oscillations.

Femtosecond time-resolved transient absorption spectroscopy was performed for a nonfluorescent solvatochromic dye, phenol blue, N-(4-dimethylaminophenyl)-1,4-benzoquinoneimine, which exhibits ultrafast nonradiative decay due to its flexible molecular structure. By exciting the molecule in ethanol (EtOH) solution with two excitation wavelengths located at shorter- and longer-wavelength sides of the visible absorption band, we observed ultrafast nonradiative decay from the excited state, followed by spectral evolution in the ground state. The nonradiative decay in the subpicosecond range creates a vibrationally hot ground state with the lifetime in the picosecond range. Subsequently, a tautomer that absorbs at shorter wavelengths is produced from the hot state, which causes a red shift of the ground-state bleach (GSB). The tautomerization presumably involves twisting of the benzoquinoneimine moiety induced by the breaking of the hydrogen bond (H-bond) between the solute and the solvent molecules. The recombination of the H-bond occurs with a time constant of ∼30 ps, and the system returns to its original state. We also observed low-frequency coherent wavepacket oscillations that modulate the GSB with dephasing times similar to the excited-state lifetime.

Multiphoton-gated cycloreversion reactions of photochromic diarylethene derivatives with low reaction yields upon one-photon visible excitation.

Cycloreversion processes of three photochromic diarylethene derivatives with extremely low one-photon reaction yields (5.0 x 10(-5) to 1.5 x 10(-2)) were investigated by means of femtosecond and picosecond laser photolysis methods. Femtosecond visible laser photolysis revealed that the excited state of the closed form in these three derivatives decayed into the ground state with 0.7-8 ps time constants and with low cycloreversion yields that were consistent with those obtained by steady-state light irradiation. On the other hand, the cycloreversion reaction was drastically enhanced by picosecond 532 nm laser excitation for all of the three derivatives. From excitation intensity effects of the reaction yield and dynamic behavior, it was found that the successive two-photon absorption process leading to higher excited states opened an efficient cycloreversion channel, with reaction yields of 0.3-0.5. These results are discussed from the viewpoint of the one-photon inerasable but two-photon erasable photochromic system.

Spectroscopic Signature of the Steric Strains in an Escherichia coli RNase HI Cavity-Filling Destabilized Mutant Protein.

A cavity-filling mutation at a hydrophobic cavity is a useful method for increasing protein stability. This method, however, sometimes destabilizes the protein because of the accompanying structural changes by the steric hindrance around the cavity. Thus, detailed knowledge of unfavorable structural changes is important for a comprehensive understanding of the cavity-filling mutation. In the present study, by employing the cavity-filling mutant of Escherichia coli RNase HI as a case study, the structural change induced by the substitution of Phe for Ala52 (Ala52Phe) was analyzed in detail using Raman spectroscopy. In previous studies, the thermodynamic result apparently indicated a small decrease in ΔG (destabilization) by the mutation. In the present study, Raman differential spectra show a clear structural difference between wild-type E. coli RNase HI and Ala52Phe. Consequently, the direct signature of the conformational strains around the protein cavity is readily acquired, leading to further understanding of the trade-off relationship between the cavity-filling and incidental steric hindrance.

Energy transfer at heterogeneous protein-protein interfaces to investigate the molecular behaviour in the crowding environment.

Investigation of the behaviour of proteins in crowded environments is crucial for understanding the role of proteins in biological environments. In this study, the behaviour of bovine serum albumin (BSA) in crowded (highly concentrated) environments was investigated using time-resolved fluorescence spectroscopy as a model system. By using energy transfer as a molecular ruler, the crowding effect was clearly observed in the time resolved spectra. In addition, by using both time resolved anisotropy measurement and Raman spectroscopy, more detail insights from conformational and dynamic points of view were described. Consequently, it was revealed that in the highly concentrated solution, most of the BSA molecules are in the fast-reversible oligomeric state and the association at the "hard" and "soft" interfaces between protein surfaces occurred in a highly crowded environment with the aid of a charge-charge and short-range attractive interface. From both the conformational and dynamic aspects, the detail spectroscopic understanding of the behaviour of BSA in the crowding environment was obtained.

Application of Raman spectroscopy for visualizing biochemical changes during peripheral nerve injury in vitro and in vivo.

Raman spectroscopy can be used for analysis of objects by detecting the vibrational spectrum using label-free methods. This imaging method was applied to analysis of peripheral nerve regeneration by examining the sciatic nerve in vitro and in vivo. Raman spectra of intact nerve tissue had three particularly important peaks in the range 2800-3000 cm-1. Spectra of injured sciatic nerves showed significant changes in the ratio of these peaks. Analysis of cellular spectra suggested that the spectrum for sciatic nerve tissue reflects the axon and myelin components of this tissue. Immunohistochemical analysis showed that the number of axons and the myelinated area were reduced at 7 days after injury and then increased by 28 days. The relative change in the axon to myelin ratio showed a similar initial increase, followed by a decrease at 28 days after injury. These changes correlated with the band intensity ratio and the changes in distribution of axon and myelin in Raman spectral analysis. Thus, our results suggest that label-free biochemical imaging with Raman spectroscopy can be used to detect turnover of axon and myelin in peripheral nerve regeneration.