UV CD Spectra Research Articles

Binding mechanism, photo-induced cleavage and computational studies of interaction cefepime drug with Human serum albumin

The binding interaction of cefepime to human serum albumin (HSA) in aqueous solution was investigated by molecular spectroscopy (UV spectra, fluorescence spectra and CD spectra), photo-cleavage and modeling studies under simulative physiological conditions. Spectrophotometric results are rationalized in terms of a static quenching process and binding constant (Kb ) and the number of binding sites (n ≈ 1) were calculated using fluorescence quenching approaches at three temperature settings. Thermodynamic data of ΔG, ΔH and ΔS at different temperatures were evaluated. The results showed that the electrostatic and hydrogen bonding interactions play a major role in the binding of cefepime to HSA. The value of 3.4 nm for the distance r between the donor (HSA) and acceptor (cefepime) was derived from the fluorescence resonance energy transfer (FRET). FTIR and CD measurements has been reaffirmed HSA–cefepime association and demonstrated reduction in α-helical content of HSA. Furthermore, the study of molecular modeling also indicated that cefepime could strongly bind to the site I (subdomain IIA) of HSA. Additionally, cefepime shows efficient photo- cleavage of HSA cleavage. Our results may provide valuable information to understand the pharmacological profile of cefepime drug delivery in blood stream. Communicated by Ramaswamy H. Sarma

Diosmin: A Daboia russelii venom PLA2s inhibitor- purified, and characterized from Oxalis corniculata L medicinal plant

Ethnopharmacological relevanceOxalis corniculata L is a medicinal plant that belongs to the Oxalidaceae family. It is a little, slow-growing plant with a frail appearance typically found in mild temperate and tropical areas like Pakistan and India. This plant also includes many other bioactive substances, including alkaloids, flavonoids, terpenoids, cardiac glycosides, saponins, phlobatannins, and steroids. Aim of the studyTo investigate the anti-inflammatory effects of Compound diosmin, which is derived from Oxalis corniculata L, on VRV-PL-5 and VRV-PL-8a isolated from Vipera russelli. Materials and methodsExtraction, purification, and characterization of bioactive by TLC, HPTLC, FT-IR analysis, UV–Vis spectrophotometer, LC-MS/MS Analysis, NMR, XRD Analysis, In vitro evaluation, Circular dichroism spectroscopy, in vivo, and in silico studies. ResultsIn this study, the extract of Oxalis corniculata was evaluated for its in vitro and in vivo anti-inflammatory effect against PLA2. The methanolic extract decreased hemolytic activity by about 60% at 1:75 w/w and neutralized the hemolytic activity completely at 1:100 w/w concentration. Diosmin inhibited VRV-PL-5 and VRV-PL-8a in a dose-dependent manner, with the extent of inhibition being about 56% for VRV-PL-5120 μM and VRV-PL-8a by 62% at the same concentration with IC50 concentrations of 87.08 μM for VRV-PL-5 and 82.08 μM for VRV-PL-8a, while at 75 μM. Diosmin inhibited the hemolytic activity of VRV-PL-5 by about 85%, and at the same concentration, VRV-PL-8a inhibited by about 75%. UV-CD spectra at the IC50 concentration of diosmin disrupted the secondary structure of VRV-PL-5 &VRV-PL-8a. In vivo, studies showed decreased myotoxicity and cardiotoxicity of the VRV-PL-5 &VRV-PL-8a, which was seen in the decrease in cytoplasmic markers LDH and CPK levels in the serum when incubated with diosmin. Furthermore, Histopathological studies of Muscles and lungs revealed that diosmin considerably protects against cellular abnormality caused by VRV-PL-5 & VRV-PL-8a. Molecular docking, MM/GBSA, and molecular dynamics simulation studies show that the diosmin is a potent inhibitor for VRV-PL-5 and VRV-PL-8a. ConclusionThis study shows that diosmin is a potentially effective VRV-PL-5 and VRV-PL-8a.

Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol

Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli. The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.

Corosolic Acid Inhibits Secretory Phospholipase A2IIa as an Anti-Inflammatory Function and Exhibits Anti-Tumor Activity in Ehrlich Ascites Carcinoma Bearing Mice.

Inflammation is generally connected to tumour progression and development. The secretory phospholipase A2IIa (sPLA2IIa) is an important inflammatory enzyme that catalyse the hydrolysis of membrane phospholipids into arachidonic and lysophosphatidic acid, which are the precursors for production of a lot of pro-inflammatory mediators like prostaglandins, prostacyclins, thromboxanes, leukotrienes and platelet activating factors, which involved in the proliferation, migration, invasion, and metastasis. Therefore, investigating safe and effective sPLA2IIa inhibitors as a therapeutic agent to treat cancer is indeed in need. Anti-inflammatory function of corosolic acid was evaluated by docking it with sPLA2IIa enzyme, sPLA2IIa inhibition, calcium and substrate concentration-dependent assays; intrinsic fluorescence and UV-CD analysis; neutralisation of sPLA2IIa induced indirect hemolytic and edema. Evaluated the anticancer activity of corosolic acid by MTT assays and caspase-3 expression; the anti-tumour activity by EAC-induced cell line and interleukin 6 expression. The corosolic acid inhibits sPLA2IIa activity to 82.21±2.82%. The inhibition was evaluated by increasing calcium from 2.5 to 15 µM and substrate from 20 to 120 nM, it did not affect the level of inhibition. Corosolic acid altered the intrinsic fluorescence and UV-CD spectra of sPLA2IIa enzyme, indicating the direct interaction. It neutralised sPLA2IIa induced hemolytic activity from 97±1.23% to 15.75±1.44% and edema from 171.51±2.39% to 119.3±2.6%. Further, as antiproliferative activity, corosolic acid reduced the PC3 cell viability from 99.66±0.57% to 23±2.64% and suppressed LPS-induced IL-6 level from 94.35±2.2% to 34.36±2.4%. It increased mean survivability time from 30 to 38 days and displayed the drug-like qualities. All the experimental results have proven the corosolic acid as an anti-inflammatory and anticancer molecule that may further be used to develop it as a drug.

Characterization of the binding between anti-tumor drug 5-fluorouracil and human alpha-2-macroglobulin: spectroscopic and molecular docking analyses

5-Fluorouracil (5-FU) is a well-recognized anticancer drug used in the treatment of tumors of head, neck and breast. Drug pharmacokinetics is affected upon binding with protein, thus, making drug–protein interactions imperative to study. Present work investigates the interaction between 5-FU and human major antiproteinase-alpha-2-macroglobulin (α2M) by multi-spectroscopic, calorimetric and molecular docking techniques. UV/Visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods have been employed to unveil the mode and mechanism of 5-FU-α2M interaction. Synchronous fluorescence showed alteration in the microenvironment of tryptophan and tyrosine residues of protein. Far UV-CD spectra suggest slight alterations in the secondary structure of α2M by 5-FU. Thermodynamic parameters determined by fluorescence quenching experiments and isothermal titration calorimetry (ITC) suggested the involvement of hydrogen bonds and hydrophobic interactions. Moreover, ITC corroborate the spontaneous and exothermic nature of the interaction process. Molecular docking illustrates that 5-FU binds with moderate affinity and Asp953, Tyr1264, Lys1236, Thr1232, Tyr1323 and Leu951 were the main residues involved. Molecular dynamics simulation studies suggested that 5-FU was stabilizing the α2M structure and forming a stable complex. It was concluded that 5-FU lower the antiproteolytic activity of α2M significantly and causes disruption in the native structure and conformation of α2M. Communicated by Ramaswamy H. Sarma

Insight into the binding of PEG-400 with eye protein alpha-crystallin: Multi spectroscopic and computational approach: possible therapeutics targeting eye diseases

Alpha-crystallin (α-crystallin) is an important eye protein having chaperone activity; its aggregation and precipitation are vital in cataract development. Polyethylene glycol-400 (PEG-400) is an important constituent of eye drops and artificial tears. The present study was targeted to study the binding of α-crystallin and PEG-400 employing multi spectroscopy, isothermal titration calorimetry (ITC) along with molecular modeling and docking approach. There was an apparent hypochromism in α-crystallin in the presence of varying PEG-400 concentrations; the binding constant obtained was 0.9 X 105 M−1 implying that strong binding is taking place between α-crystallin and PEG-400. Fluorescence spectroscopy suggested good binding of PEG-400 to α-crystallin with a binding constant (K) of 106 M−1. Moreover, fluorescence quenching studies carried out at three different temperatures suggested α-crystallin-PEG-400 complex formation to be guided by combination of static and dynamic modes. Thermodynamic parameters suggested α-crystallin-PEG-400 complex formation is driven by van der Waals forces and hydrogen bonding, making it seemingly specific. Far UV-CD spectra revealed no shift in the peak implying no alterations in the secondary structure of α-crystallin upon PEG-400 binding further validating complex formation. In vitro assays were further entrenched by in silico assays. Molecular modeling was used to make the functionally active form of α-crystallin. A binding pocket located in the β chain was delineated by Prank Web; molecular docking showed binding of PEG-400 in this pocket. This study will give an insight into the binding of PEG-400 with α-crystallin and can serve as a rationale for the discovery of therapeutic molecules that can be used for the treatment of eye-related crystallin-directed diseases. Highlights PEG-400 is a constituent of lubricant eye drops. Molecular modelling gave functionally active crystallin. Molecular docking showed PEG-400 in the binding pocket. Fluorescence binding revealed good bindingα-crystallin and PEG400. The α-crystallin-PEG-400 complex was guided by static and dynamic quenching. Communicated by Ramaswamy H. Sarma

Comprehensive insight into the molecular interaction of an anticancer drug-ifosfamide with human alpha-2-macroglobulin: biophysical and in silico studies

Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein, making the studies on drug-protein interaction promising. The present study investigates the interaction between ifosfamide and human antiproteinase-alpha-2-macroglobulin (α2M) by using multi-spectroscopic and in silico techniques. The UV-visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods were employed to unveil the mode and mechanism of ifosfamide–α2M interaction. Fluorescence quenching studies performed at three different temperatures indicated that ifosfamide–α2M complex formation involves static quenching. Far UV-CD spectra revealed a minor alteration in the secondary structure of α2M instigated by ifosfamide. The thermodynamic parameters determined by fluorescence quenching experiment and isothermal titration calorimetry (ITC) suggested that the complex between ifosfamide and α2M involves hydrogen bonding and hydrophobic interactions. Molecular docking illustrates that ifosfamide binds with moderate affinity to Lys1240, Asn173, Ser957, Leu955, Asp953, Lys1216 and Thr1236 residues during the interaction. Molecular dynamic (MD) simulation suggested that the ifosfamide forms a stable complex with α2M. Communicated by Ramaswamy H. Sarma

The interaction of sodium dodecyl sulfate with trypsin: Multi-spectroscopic analysis, molecular docking, and molecular dynamics simulation

The bioactivity of enzymes is sensitive to certain factors in their application environment, such as the pH, temperature, ionic strength, and additives, which can alter the native conformation of enzymes. To determine the mechanism by which the interaction of SDS influences the structure and activity of trypsin, molecular docking, molecular dynamics simulations, DSC, and multi-spectroscopic measures including UV absorption, fluorescence, and circular dichroism were used. The results show that the hydrolytic activity towards casein could be dramatically restrained by SDS. UV absorption, CD, and fluorescence spectra demonstrated the formation of a trypsin–SDS complex. Thermodynamic parameters and molecular docking data revealed that the binding process was spontaneous, and that the main binding forces between SDS and trypsin were hydrogen bonds and van der Waals forces. In addition, molecular docking predicted that the binding site of SDS on trypsin was located at the active center. Molecular dynamic simulations showed that treatment with SDS resulted in the structure of trypsin becoming unstable and unfolded near its active center. This work provides insights into the interaction of SDS with trypsin on the molecular level and is beneficial to understanding of how SDS affects the conformation and activity of trypsin in application processes.

The effect of magnetized water on the oxidation reaction of phenol derivatives and aromatic amines by horseradish peroxidase enzyme.

The present study aimed to investigate, for the first time, the rate of the oxidation reaction of some derivatives of phenol and aromatic amines, that is, pyrogallol, catechol, resorcinol, ortho-aminophenol, meta-aminophenol, para-aminophenol, ortho-phenylenediamine, and para-phenylenediamine, in the presence of hydrogen peroxide in pure and magnetized solvents using horseradish peroxidase enzyme. The reaction was studied in the absence and presence of a magnetized solvent under completely identical conditions. The results showed that magnetized solvent could change the structure of the enzyme and reduce its activity. In addition, it affected the rate of oxidation of the selected derivatives through altering the strength of the hydrogen bonds of the system. The changes in the structure and activity of the enzyme were examined using UV-Vis and fluorescence spectroscopy as well as viscosity measurement technique. Examination of the secondary structure via the far UV-CD spectrum indicated the increase in the alpha helical structure in the magnetized solvent. When dissolved in a magnetized solvent, hydrogen peroxide as an enzyme substrate reduced the rate of enzymatic reaction and provided lower saturation conditions for the enzyme compared with when it was dissolved in the pure solvent.

Understanding the binding between Rosmarinic acid and serum albumin: In vitro and in silico insight

Rosmarinic acid (RA) is a natural product that is increasingly being used in food industries and cosmetic industries. Drug pharmacokinetics is affected upon binding with protein, thus making drug-protein interactions imperative to study. The binding affinity between RA and serum albumin, human serum albumin (HSA) was investigated using multi spectroscopic approaches and in silico analysis. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies were employed to elucidate the mode and the mechanism of HSA-RA interaction. Fluorescence studies showed excellent binding between HSA and RA with a binding constant (K) of 107 M−1. Fluorescence quenching experiments carried out at three different temperatures suggested that HSA-RA complex formation is guided by static quenching. The binding constants were found to decrease at higher temperatures suggesting the formation of the less stable complex at higher temperatures. Far UV-CD spectra revealed slight alterations in the secondary structure of HSA upon RA binding further validating complex formation between HSA and RA. Thermodynamic parameters obtained suggested hydrophobic interactions to play a dominant role in this interaction. Isothermal titration calorimetry (ITC) further validated the spontaneous and exothermic nature of this reaction. Molecular docking study shows that RA is binding with appreciable affinity showing specific interactions towards the binding pocket residues of HSA mimicking the binding pose of co-crystallized myristic acid. Molecular dynamics simulation study suggested that RA is stabilizing the HSA structure and leads to fewer conformational changes upon binding.

Probing the interaction of Rivastigmine Tartrate, an important Alzheimer's drug, with serum albumin: Attempting treatment of Alzheimer's disease

The present study was aimed at investigating the binding between an important drug of Alzheimer's therapy, Rivastigmine tartrate (RT), with Bovine serum albumin (BSA). BSA is a model protein that is increasingly being used for studies related to drug-protein interaction owing to its structural similarity with human serum albumin (HSA) which is extremely abundant in the circulatory system comprising around 60% of the total plasma protein. Fluorescence spectroscopy implied that complex formation is taking place between BSA and RT; binding constant calculated was of the order of 104 M−1 implicative of the strength of this interaction. Fluorescence spectroscopy was carried out at three different temperatures in a bid to find out the operative mode of quenching; static quenching was taking place for RT-BSA interaction with a binding constant of 2.5 × 104 M−1 at 298 K. Further, changes in Far UV CD spectra clearly implied that RT induces structural transition in BSA suggestive of RT-BSA complex formation. The negative value of ∆G0 as obtained from fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggests the reaction to be spontaneous and thermodynamically favorable. Additionally, molecular docking was employed to investigate different forces and critical residues involved in RT-BSA interaction. Furthermore, all-atom molecular dynamics simulation for 50 ns was performed on the BSA-RT complex to investigate its conformational behavior, stability and dynamics.

Understanding the binding interaction between methotrexate and human alpha-2-macroglobulin: Multi-spectroscopic and computational investigation

Methotrexate (MTX) is advised in the treatment of solid tumours, hematologic malignancies and autoimmune disorders. On reaching the circulation, 60% of MTX is bound to the proteins present in serum. Alpha-2-macroglobulin (α2M) is a plasma proteinase inhibitor with numerous functions such as binding, transportation and targeting of molecules. Our studies are the first attempt to investigate the binding interaction of pharmacologically important drug MTX, and highly abundant proteinase inhibitor- α2M. The protein functional activity assay shows 53% decrease in antiproteolytic potential of α2M upon drug interaction. The binding of MTX with α2M was studied by various biophysical methods. UV–visible absorption spectroscopy reveals hyperchromicity of α2M spectra upon drug binding. The intrinsic fluorescence spectra show quenching in fluorescence intensity of α2M and the mechanism of quenching was found to be static in nature. Far UV-CD spectra unveil slight alteration in secondary structure of α2M upon drug binding. Isothermal titration calorimetry (ITC) reveals the value of thermodynamic parameters and which affirms the binding process to be spontaneous and exothermic. Molecular docking illustrates that Asn173, Leu1298, Gly172, Lys1240, Gln1325, Ser1327, Glu913, Asn1139, Lys1236, Leu951 and Arg1297 were the key residues involved during interaction process. Molecular dynamics (MD) simulation studies suggest that MTX form a stable complex with α2M. Our study assumes importance from the fact that MTX is known to bind plasma proteins quite efficiently.

Application of spectral methods for studying DNA damage induced by gamma-radiation

Spectral methods can provide a variety of possibilities to determine several types of radiation-induced DNA damage, such as nucleobase destruction and local denaturation. DNA UV absorption and CD spectra measured at room temperature undergo noticeable alteration under the action of γ-radiation. We have applied the Spirin method of total nucleobases determination, and have measured the molar extinction coefficient of DNA and DNA CD spectra for solutions with different NaCl concentrations (3mM–3.2M) and containing MgCl2, exposed to γ-radiation with the doses of 0–103Gy. The melting temperatures of DNA in irradiated solutions at the doses of 0–50Gy were obtained with the help of spectrophotometric melting. It was found that the amount of destructed nucleobases and radiation-induced loss of DNA helicity significantly decreases with the rise of the ionic strength of the irradiated solution. Substitution of a portion of Na+ ions on Mg2+ while keeping the total ionic strength constant (μ=5mM) does not affect the considered radiation effects. The role of the structure and composition of the DNA secondary hydration layer in the radiation-induced damages is discussed.

Thermal stability and conformation of DNA and proteins under the confined condition in the matrix of hydrogels.

Spatially confined environments are seen in biological systems and in the fields of biotechnology and nanotechnology. The confinement restricts the conformational space of polymeric molecules and increasing the degree of molecular crowding. Here, we developed preparation methods for agarose and polyacrylamide gels applicable to UV spectroscopy that can evaluate the confinement effects on DNA and protein structures. Measurements of UV absorbance and CD spectra showed no significant effect of the confinement in the porous media of agarose gels on the base-pair stability of DNA polynucleotides [poly(dA)/poly(dT)] and oligonucleotides (hairpin, duplex, and triplex structures). On the other hand, a highly confined environment created by polyacrylamide gels at high concentrations increased the stability of polynucleotides while leaving that of oligonucleotides unaffected. The changes in the base-pair stability of the polynucleotides were accompanied by the perturbation of the helical conformation. The polyacrylamide gels prepared in this study were also used for the studies on proteins (lysozyme, bovine serum albumin, and myoglobin). The effects on the proteins were different from the effects on DNA structures, suggesting different nature of interactions within the gel. The experimental methods and results are useful to understand the physical properties of nucleic acids and proteins under confined conditions.

Role of Cysteine Residues in Regulation of Peptidyl-prolyl cis-trans Isomerase Activity of Wheat Cyclophilin TaCYPA-1.

The wheat cyclophilin, TaCYPA-1, shows peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, the significance of different cysteine residues in regulation of PPIase activity of this protein has not been investigated. The main objective of this study was to analyze the role of different disulphide linkages in redox mechanisms that modulate the PPIase activity of TaCYPA-1. Site-directed mutants of TaCYPA-1 were generated by substituting cysteine residues at positions -40 and -122 with serine and -126 with proline. The recombinant proteins were expressed in Escherichia coli and purified. The effect of Cu2+ and N-ethylmaleimide was studied on PPIase activity of the purified recombinant cyclophilins for analyzing the role of different cysteine residues in the modulation of enzyme activity. The changes in secondary structure of TaCYPA-1 and its mutants were analysed by recording far UV CD spectra. The effect of different cysteine substitutions on thermotolerance of E. coli was studied by monitoring the cell growth at 47 °C after 2 h, 3 h and 5 h of heat stress. The catalytic efficiencies (Kcat/Km) of TaCYPA-1C40S (0.37 X 106 M-1 s-1) and TaCYPA- 1C122S (0.31 X 106 M-1 s-1) were significantly lower as compared to the native TaCYPA-1 (1.33 X 106 M-1 s-1), whereas Kcat/Km of the double mutant TaCYPA-1C40S/C122S was significantly higher (2.36 X 106 M-1 s-1). Compared to the wild-type TaCYPA-1, the different mutants also showed differential sensitivity to Cu2+. Furthermore, the results of this study also revealed that despite lacking PPIase activity, the mutant TaCYPA-1C126P was able to confer partial protection against heat stress. This study revealed that mutation of different cysteine residues in TaCYPA-1 results in differential effect on PPIase activity. It was also observed that of the two pairs of cysteine residues i.e. Cys40/Cys168 and Cys122/Cys126, the latter appears to play major role in redox regulation of TaCYPA-1. The mutant TaCYPA-1C126P, though lacking PPIase activity, was able to impart partial tolerance to heat stress in E. coli, suggesting other functions besides cis to trans isomerisation. This study, therefore, provides new insights into the regulation of plant cyclophilins.

The presence of non-native helical structure in the unfolding of a beta-sheet protein MPT63.

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β-sandwich protein containing 11 β strands and a very short stretch of 310 helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β-sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV-CD and FTIR spectra clearly suggest that the low-pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β-sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non-native helical propensity of the protein structure.

Inhibition of Aggregation in B-Sheet Model Peptide by PPII Helix Capping

A 12mer peptide (Beta, SWTVEGNKYTYK-NH2) was designed based on the Trpzip model. Beta can form an aggregate with fibril like morphology imaged by TEM. The secondary structure of the fibril was characterized to be anti-parallel β-sheets by FT-IR spectra. VCD (Vibrational Circular Dichroism) spectrum demonstrated the supramolecular chirality of the fibril and UV-CD spectrum of this peptide confirmed the tertiary contact between aromatic residues in the sequence. Upon mutation with an added PPII helix inducing combo to the C-terminal of this peptide, a new peptide BP (SWTVEGNKYTYKNGAPPPK-NH2) was synthesized. BP did not enhance ThT fluorescence, its secondary structure was characterized to be unordered by UV-CD and FT-IR spectra. Further FRET analysis with a dansylated version of BP shows that the end to end distance is consistent with the proline mutations disrupting the aromatic contacts and thus stabilized the unordered conformation. In contrast, 1:1 ratio mixture of a 9mer PPII conformation peptide and Beta did not change the aggregation behavior of Beta. This study points out that sequence mutation is crucial in order to change the propensity of aggregation in peptides.

Sub-Micellar Concentration of Sodium Dodecyl Sulphate Prevents Thermal Denaturation Induced Aggregation of Plant Lectin, Jacalin.

The irreversible thermal unfolding of jacalin, the lectin purified from jackfruit seeds was accompanied by aggregation, where intermolecular interactions among the subunits are favoured over intramolecular interactions. The extent of aggregation increased as a function of temperature, time and protein concentration. The anionic surfactant, sodium dodecyl sulphate (SDS) significantly suppressed the formation of aggregates as observed by turbidity measurements and Rayleigh scattering assay. Moreover, far UV-CD spectra indicate that the protein β sheet transforms into α helical structure, when denatured in the presence of 3mM SDS. Further, jacalin when heated in the presence of SDS partially retained the hemagglutination activity when jacalin-SDS mixture was diluted to 1:8 factor since 3mM SDS was found to lyse the red blood cells. Thus, SDS only altered the aggregation behaviour of jacalin by preventing intermolecular hydrogen bonding among the exposed residues but did not completely stabilize the native conformation.

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 Kb 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.

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.