The link between restriction endonuclease fidelity and oligomeric state: A study with Bse634I

Type II restriction endonucleases (REases) exist in multiple oligomeric forms. The tetrameric REases have two DNA binding interfaces and must synapse two recognition sites to achieve cleavage. It was hypothesised that binding of two recognition sites by tetrameric enzymes contributes to their fidelity. Here, we experimentally determined the fidelity for Bse634I REase in different oligomeric states. Surprisingly, we find that tetramerisation does not increase REase fidelity in comparison to the dimeric variant. Instead, an inherent ability to act concertedly at two sites provides tetrameric REase with a safety-catch to prevent host DNA cleavage if a single unmodified site becomes available.     

Explication of bovine serum albumin binding with naphthyl hydroxamic acids using a multispectroscopic and molecular docking approach along with its antioxidant activity

In the present investigation, the protein‐binding properties of naphthyl‐based hydroxamic acids (HAs), N‐1‐naphthyllaurohydroxamic acid ( 1 ) and N‐1‐naphthyl‐p‐methylbenzohydroxamic acid ( 2 ) were studied using bovine serum albumin (BSA) and UV–visible spectroscopy, fluorescence spectroscopy, diffuse reflectance spectroscopy–Fourier transform infrared (DRS–FTIR), circular dichroism (CD), and cyclic voltammetry along with computational approaches, i.e. molecular docking. Alteration in the antioxidant activities of compound 1 and compound 2 during interaction with BSA was also studied. From the fluorescence studies, thermodynamic parameters such as Gibb's free energy (ΔG), entropy change (ΔS) and enthalpy change (ΔH) were calculated at five different temperatures (viz., 298, 303, 308, 313 or 318 K) for the HAs–BSA interaction. The results suggested that the binding process was enthalpy driven with dominating hydrogen bonds and van der Waals’ interactions for both compounds. Warfarin (WF) and ibuprofen (IB) were used for competitive site‐specific marker binding interaction and revealed that compound 1 and compound 2 were located in subdomain IIA (Sudlow's site I) on the BSA molecule. Conclusions based on above‐applied techniques signify that various non‐covalent forces were involved during the HAs–BSA interaction. Therefore the resulted HAs–BSA interaction manifested its effect in transportation, distribution and metabolism for the drug in the blood circulation system, therefore establishing HAs as a drug‐like molecule.     

Interaction of Prussian blue nanoparticles with bovine serum albumin: a multi-spectroscopic approach

Owning to their exceptional properties, Prussian blue nanoparticles (PBNPs) are promising in a variety of biomedical applications. In this scenario, understanding of how PBNPs interact and behave in biological systems is essential. Herein, the interaction of PBNPs with protein was investigated. Specifically, the citric acid stabilized PBNPs with a size of 10 nm were synthesized and characterized. The interactions of these PBNPs with the model protein, bovine serum albumin (BSA), were then probed by spectroscopic methods. It was found that the BSA intrinsic fluorescence was quenched upon addition of PBNPs due to the static interaction, suggesting the binding of PBNPs with BSA. Moreover, the synchronous fluorescence and circular dichroism spectra indicated the conformational change of BSA due to the presence of PBNPs.     

Correlation between Shiga toxin B-subunit stability and antigen crosspresentation: a mutational analysis

The homopentameric B-subunit of Shiga toxin (STxB) is used as a tool to deliver antigenic peptides and proteins to the cytosolic compartment of dendritic cells (DCs). In this study, a series of interface mutants of STxB has been constructed. All mutants retained their overall conformation, while a loss in thermal stability was observed. This effect was even more pronounced in trifluoroethanol solutions that mimic the membrane environment. Despite this, all mutants were equally efficient at delivering a model antigenic protein into the MHC class I-restricted antigen presentation pathway of mouse DCs, suggesting that the structural stability of STxB is not a key factor in the membrane translocation process.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。     

The mechanical role of VASP in an Arp2/3-complex-based motility assay

The comet motility assay, inspired by Listeria locomotion, has been used extensively as an in vitro model to study the structural and motile properties of the actin cytoskeleton. However, there are no quantitative measurements of the mechanical properties of these actin comets. In this work, we use nanoindentation based on atomic force microscopy to measure the elastic modulus of actin comets grown on  1-μm-diameter beads in an Arp2/3 (actin-related proteins 2 and 3)-complex-dependent fashion in the absence and in the presence of VASP (vasodilator-stimulated phosphoprotein). Recruitment of VASP to the bead surface had no effect on the initial velocity or morphology of the comets. Instead, we observed an improved contact of the comets with the beads and an increased elastic modulus of the comets. The VASP-mediated increase in elastic modulus was dependent on both concentration and ionic strength. In conclusion, we propose that VASP plays a mechanical role in Arp2/3-complex-dependent motility by amplifying the elastic modulus of the thus assembled actin network and, consequently, by strengthening its cohesion for persistent protrusion.     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Exploring the interaction of the photodynamic therapeutic agent thionine with bovine serum albumin: multispectroscopic and molecular docking studies

This study explores the binding interaction of thionine (TH) with bovine serum albumin (BSA) under physiological conditions (pH 7.40) using absorption, emission, synchronous emission, circular dichroism (CD) and three‐dimensional (3D) emission spectral studies. The results of emission titration experiments revealed that TH strongly quenches the intrinsic emission of BSA via a static quenching mechanism. The apparent binding constant (K) and number of binding sites (n) were calculated as 2.09 × 10⁵ dm³/mol and n~1, respectively. The negative free energy change value for the BSA–TH system suggested that the binding interaction was spontaneous and energetically favourable. The results from absorption, synchronous emission, CD and 3D emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure in BSA. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of BSA. The molecular docking study further substantiates Sudlow site I as the preferable binding site of TH in BSA. Copyright © 2014 John Wiley & Sons, Ltd.     

Spectroscopic exploration and thermodynamic characterization of desvenlafaxine interacting with fluorescent bovine serum albumin

The mechanism of the interaction between bovine serum albumin (BSA) and desvenlafaxine was studied using fluorescence, ultraviolet absorption, 3‐dimensional fluorescence spectroscopy, circular dichroism, synchronous fluorescence spectroscopy, cyclic voltametry, differential scanning calorimetry, and attenuated total reflection–Fourier transform infrared spectroscopic techniques under physiological condition at pH 7.4. Stern‐Volmer calculations authenticate the fluorescence of BSA that was quenched by desvenlafaxine in a collision quenching mode. The fluorescence quenching method was used to evaluate number of binding sites “n” and binding constant K _A that were measured, and various thermodynamic parameters were evaluated at different temperatures by using the van't Hoff equation and differential scanning calorimetry technique, which indicated a spontaneous and hydrophobic interaction between BSA and desvenlafaxine. According to the Förster theory we calculate the distance between the donor, BSA and acceptor, desvenlafaxine molecules. Furthermore, circular dichroism and attenuated total reflection–Fourier transform infrared spectroscopy indicate nominal changes in the secondary structure of the protein.     

Molecular interactions of MnO2@RGO (manganese dioxide-reduced graphene oxide) nanocomposites with bovine serum albumin

Abstract

Graphene based materials have attracted global attention due to their excellent properties. GO-metal oxide nanocomposites have been conjugated with biomolecules for the development of novel materials and potentially used as biomarkers. Herein, a detailed study on the interaction of Bovine serum albumin (BSA) with MnO₂@RGO (manganese dioxide-reduced graphene oxide) nanocomposites (NC) has been carried out. MnO₂@RGO nanocomposites were prepared through a template/surfactant free hydrothermal route at 180?°C for 12?h by varying the graphene oxide (GO) concentration. Different biophysical experiments have been carried out to evaluate molecular interactions between BSA and NCs. Intrinsic fluorescence has been used to quantify the quenching efficiency of NCs and the binding association of BSA-NC complexes. NCs effectively quenched the intrinsic fluorescence of BSA via static and dynamic mechanism. Further, the results indicate that the molecular interactions of NC with BSA are dependent on the GO percentage in NC. Circular dichroism results demonstrate nominal changes in the secondary structure of BSA in presence of NCs. Also, the esterase-like activity of BSA was marginally affected after adsorption upon NCs. In addition, the FESEM micrographs reveal that the protein-NC complexes consist of nanorod and sheet-like morphologies are forming aggregates of different sizes. We hope that this study will provide a basis for the design of novel graphene based and other related nanomaterials for several biological applications.

Communicated by Ramaswamy H. Sarma     

Binding of anti-cardiovascular drug to serum albumin: an insight in the light of spectroscopic and computational approaches

Isoprenaline hydrochloride is a potential cardiovascular drug helps in the smooth functioning of the heart muscles. So, we have performed the binding study of ISO with BSA. This study was investigated by UV absorption, fluorescence, synchronous fluorescence, circular dichroism, etc. The analysis of intrinsic fluorescence data showed the low binding affinity of ISO. The binding constant K_b was 2.8 × 103 M-1 and binding stoichiometry (n) was approximately one and the Gibb’s free energy change at 310 K was determined to be -8.69 kcal mol^?1. Negative Gibb’s free energy change shows the spontaneity of the BSA and ISO interaction. We have found ISO-induced alternation in the UV absorption, synchronous fluorescence and CD spectra in the absence and presence of the quencher indicates the complex formation. In synchronous fluorescence, red shift was obtained because of the complex formation of BSA and ISO. The distance (r) between the BSA (donor) and ISO (acceptor) was 2.89 nm, determined by FRET. DLS measurements interpreted complex formation due to the reduction in hydrodynamic radii of the protein in the presence of the drug. The binding site of ISO was found to be nearer to Trp 134 with the help of molecular docking and the ΔG° was found to be –10.2 kcal mol^?1. The esterase activity result suggests that ISO acts as competitive inhibitor. Thus, this study would help to determine the binding capacity of the drug to the protein which may indicate the efficiency of diffusion of ISO into the blood for the treatment of heart diseases.     

Non-random binding of N-acetoxy-N-2-acetylaminofluorene to chromatin subunits as visualized by immunoelectron microscopy

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.     

Biochemical and biophysical studies on cytochrome aa3 II. Conformations of oxidized cytochrome aa3

1.

1. The spectral properties of ‘oxygenated’ cytochrome c oxidase, prepared by passing air through the dithionite-reduced enzyme solution, were compared with those of the ferric enzyme.     

Circular dichroism studies of the complex between ferredoxin and ferredoxin-NADP reductase

Circular dichroism (CD) spectra are presented of ferredoxin, ferredoxin-NADP reductase and their complex. A change in CD occurs on complex formation which is consistent with a decrease in the Cotton effects due to the ferredoxin. This change is interpreted as due to a decrease in interaction in ferredoxin between the iron-sulphur chromophore group and the protein.     

Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.     

Study of fluorescence interaction and conformational changes of bovine serum albumin with histamine H1‐receptor–drug epinastine hydrochloride by spectroscopic and time‐resolved fluorescence methods

The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H₁ drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10⁴, 5.5 × 10⁴, and 3.9 × 10⁴M⁻¹). Based on the thermodynamic parameters (ΔH⁰, ΔG⁰, and ΔS⁰), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non‐radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV–visible, synchronous fluorescence, CD, and three‐dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 646–657, 2015.