Mohd Ishtikhar

Revisiting ligand-induced conformational changes in proteins: essence, advancements, implications and future challenges

Ligand-induced conformational changes are of immense importance for the biological activity of a protein. An in-depth understanding of salutary and deleterious effects of ligand-induced conformational alterations in single- and multi-chain proteins would lend a hand in human welfare. Unlike single-chain proteins, the function of multichain proteins depends upon the inherent properties of the subunit interfaces. The interfaces of temporary oligomeric proteins and the active sites of enzymes are of similar characteristics but the interfaces are more conservative than the active sites. Therefore, these interfaces may possibly be represented as drug targets by inhibition or induction of the oligomerization process. Thus without detailed structural understanding of ligand-induced conformational changes in a protein, structure-based rational drug designing is a great challenging task. So the purpose of this review is to clarify or enlighten the reader at the degree of internal motions related to protein backbone and side-chain flexibility which occur on binding of small molecule to a protein target. This can prove helpful to improve the conformational prediction for a protein–ligand complex. Besides a detailed description of protein–ligand interaction, this review also focuses on structure–activity relationships of protein which will surely help in the rational drug designing.

Thermal induced unfolding of human serum albumin isomers: assigning residual α helices to domain II.

In this study we have investigated the heat induced denaturation of HSA by utilizing spectroscopic approaches including fluorescence and circular dichroism. Thermal denaturation of N isomer (domains I-III remain intact), B isomer (loss of helical structure of interdomain contacts) and I state (domain II intact) was found to be co-operative processes while for F isomer domains unfold non-cooperatively. These finding pointed out that during N-F transition, HSA suffers more structural alterations which are not localized only to domain III. Loss of secondary structure in the temperature range 20-60 °C without effecting tertiary structure of N isomer of HSA is mainly due to loss in helical extensions connecting domain I to II and domain II to III. All the four thermally denatured states (60-96 °C) of HSA retained approximately 50% residual α-helical structures. Near-UV spectroscopy used as a probe for tertiary structure indicated that heat denatured states lost almost all of the tertiary contacts thereby forming molten globule like states. Furthermore, our results provide evidence that residual helical structures are mainly located in domain II.

Biophysical and molecular docking insight into interaction mechanism and thermal stability of human serum albumin isoforms with a semi-synthetic water-soluble camptothecin analog irinotecan hydrochloride

In the present work, we have examined the binding parameters, thermodynamics, and stability of human serum albumin (HSA) isoforms at pH 7.4 and 9.0, using spectroscopic, calorimetric, and molecular docking methods in the presence of water-soluble camptothecin analog irinotecan hydrochloride (CPT-11). We observed that CPT-11 binds to HSA through a static quenching procedure of ground-state complex formation with N-isoform and B-isoform. Hydrogen bond and hydrophobic interactions are the major governing forces that participating in the formation of protein–drug complex. To determine the binding site of CPT-11 within HSA molecules, we also have performed molecular docking experiments. We explored the CPT-11-mediated stability and modulation of HSA by performing dynamic light scattering (DLS) and differential scanning calorimetry (DSC) experiments. DLS and DSC techniques are used to determine the size and the melting point (Tm) of HSA, which was decreased in the presence of CPT-11. Therefore, CPT-11 plays an important role in HSA stability and protein–ligand interactions. The present study provides valuable information in the field of pharmacokinetics, pharmaco-dynamics, and drug discovery. Graphical abstract Irinotecan hydrochloride (CPT-11) binds to HSA through a static quenching mechanism by ground-state complex formation. Hydrophobic interactions and hydrogen bonds played a major role in protein–drug complex formation. The melting point (Tm) of HSA was decreased in the presence of CPT-11 as compared to native HSA that was determined by DSC for both isoforms of HSA.

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study.

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA. Graphical abstract 6-thioguanine bind to HSA through a static quenching procedure by ground-state complex formation and hydrogen bonds and hydrophobic interaction played a major role in the reaction process. GdnHCl and urea denature the HSA by two-state unfolding mechanisms, GdnHCl is more effective denaturant in comparison to urea.

Biophysical investigation of thymoquinone binding to 'N' and 'B' isoforms of human serum albumin: exploring the interaction mechanism and radical scavenging activity†

Thymoquinone (TQ) is the main constituent of Nigella sativa and is traditionally used as a folk medicine. Our aim was to investigate the binding mechanism of TQ to human serum albumin (HSA) isoforms (‘N’ form at pH 7.4 and ‘B’ form at pH 9.0) using biophysical methods such as intrinsic tryptophan fluorescence quenching, isothermal titration calorimetry (ITC), circular dichroism (CD), dynamic light scattering (DLS), Forster resonance energy transfer (FRET) and antioxidant activity in the absence and presence of TQ. We have calculated the binding and thermodynamic parameters from spectroscopic and calorimetric methods. CD and DLS were respectively used to monitor the changes in the secondary structure and hydrodynamic radii of HSA as a result of its interaction with TQ. The esterase and antioxidant or radical scavenging activities of both the isoforms of HSA were investigated in the absence/presence of TQ. The antioxidant activity of TQ was remarkably enhanced upon its interaction with HSA. Therefore, the efficiency of HSA to scavenge the free radical ions was increased in the presence of TQ which is generated in the body by various metabolic processes.

Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin

Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications.

Secondary structural changes in guanidinium hydrochloride denatured mammalian serum albumins and protective effect of small amounts of cationic gemini surfactant pentanediyl-α,ω-bis(cetyldimethylammonium bromide) and methyl-β-cyclodextrin: A spectroscopic study.

In the present study the cationic gemini surfactant assisted refolding of guanidinium hydrochloride (GdCl) denatured mammalian serum albumins viz. sheep serum albumin (SSA), rat serum albumin (RSA) and porcine serum albumin (PSA) using a combination of cationic gemini surfactants, pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N(+)-(CH2)5-N(+)(CH3)2C16H33)⋅2Br(-) designated as G5 and methyl-β-cyclodextrin in the artificial chaperone assisted two step method, is attempted. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD), and fluorescence spectroscopy. A perusal of DLS data indicates that against the native hydrodynamic radius (Rh) of 4.3nm in SSA, 3.9nm in PSA and 3.5nm in RSA, the Rh of the said proteins, when refolding is attempted by simple dilution, increases to 21.7nm, 36.6nm and 37.2nm, respectively. Hydrodynamic radii very near to the native protein, i.e., 4.0nm, 4.1nm and 4.4nm for RSA, PSA and SSA respectively, is obtained on the sequential addition of G5 and methyl-β-cyclodextrin to the denatured protein. Circular dichroism studies corroborate with the DLS data. The results obtained from the multi-technique approach are ascribed to the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in a very strong electrostatic and hydrophobic interactions. Based on the present study it is suggested that the gemini surfactants may be utilized in the protein refolding studies and thus may address one of the most pressing demand of biotechnology industry for the development of efficient and inexpensive folding aides.

Non-fluorinated cosolvents: A potent amorphous aggregate inducer of metalloproteinase-conalbumin (ovotransferrin).

Here we have used five non-fluorinated cosolvents (acetonitrile, ethanol, methanol, sec-butanol and ter-butanol) at increasing concentrations and analyzed their aggregation inducing behavior on interaction with conalbumin (CA). The aggregates were identified as amorphous by performing spectroscopic experiments like circular dichroism and dye binding assay. The amorphous aggregate contains rich β-sheet content, show insignificant increment in Thioflavin-T (ThT) fluorescence intensity but strong 1-anilino-8-napthalene sulfonate (ANS) binding with enhanced fluorescence intensity. We also performed transmission electron microscope (TEM) and scanning electron microscope (SEM) imaging of the aggregates which made the result more informative. The morphology appeared on TEM imaging shows aggregates but there is no exhibition of fibril formation, as was observed in amyloid induced by 2,2,2-trifuoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-propan-2-ol (HFIP). SEM imaging also gives the similar results indicating the formation of amorphous aggregates. Web based tools (Waltz and AGGRESCAN) predicted aggregation prone regions in CA which are accountable for the aggregation.

Inhibitory effect of copper nanoparticles on rosin modified surfactant induced aggregation of lysozyme

Protein aggregation is associated with many serious diseases including Parkinsons and Alzheimers. Protein aggregation is a primary problem related with the health of industrial workers who work with the surfactants, metal ions, and cosolvents. We have synthesized rosin-based surfactants, i.e., quaternary amines of rosin diethylaminoethyl esters (QRMAE), which is an ester of rosin acid with polyethylene glycol monomethyl ether. Here, we report the thermal aggregation of lysozyme induced by QRMAE at 65 °C and pH 7.4 for a given time period in which amorphous aggregates are formed and confirm that copper-nanoparticles have the ability to inhibit QRMAE-induced aggregation compared with zinc and silver-nanoparticles. Aggregation experiments was evaluated using several spectroscopic methods and dye binding assay, such as turbidity, Rayleigh light scattering, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) and circular dichroism (CD), that was further supported by scanning electron microscopy (SEM) and SEM with EDX. The therapeutic use of nanoparticles and the fact that rosin possesses excellent film-forming properties, and that its derivatives have pharmaceuticals application such as micro encapsulation, coating and film forming, its matrix materials are used for sustained and controlled release tablets, renders importance and application to the present study.

Temperature dependent rapid annealing effect induces amorphous aggregation of human serum albumin.

This study represents an analysis of the thermal aggregation of human serum albumin (HSA) induced by novel rosin modified compounds. The aggregation process causes conformational alterations in the secondary and tertiary structures of the proteins. The conversion of globular protein to amorphous aggregates was carried out by spectroscopic, calorimetric and microscopic techniques to investigate the factors that are responsible for the structural, conformational and morphological alteration in the protein. Our outcome results show that the aggregation of HSA was dependent on the hydrophobicity, charge and temperature, because the formation of amorphous aggregates occurs in the presence of a novel cationic rosin compound, quaternary amine of rosin diethylaminoethyl ester (QRMAE), at 40°C and pH 7.4 (but at 25°C on similar pH value, there was no evidence of aggregate formation). In addition, the parent compound of QRMAE, i.e., abietic acid, and other derivatives such as nonionic rosin compounds [(RMPEG-750) and (RMA-MPEG-750)] do not shows the aggregating property. This work provides precise and necessary information that aid in the understanding the effects of rosin derivative compounds on HSA. This study also restrains important information for athletes, health providers, pharmaceutical companies, industries, and soft drink-processing companies.