Nida Zaidi

pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “Rh” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.

Biophysical Insight into Furosemide Binding to Human Serum Albumin: A Study To Unveil Its Impaired Albumin Binding in Uremia

Exogenous substances like drugs, when absorbed, enter into the circulatory system and bind reversibly and extensively to human serum albumin (HSA). But transport of various drugs like a diuretic, furosemide (FUR), via albumin in uremia is seriously compromised due to accumulation of uremic toxins. The reason behind it is explored by investigating the binding mechanism of FUR to HSA. Isothermal titration calorimetry results show that FUR binds with HSA at high (Kb ∼ 10(4)) and low affinity (Kb ∼ 10(3)) sites whereas spectroscopic results predict binding at a single site (Kb ∼ 10(5)). Thermodynamic analysis shows that the HSA-FUR complex formation occurs via hydrogen bonds and hydrophobic interactions and undergoes slight structural changes, as evident by FTIR and far-UV CD. Further, the lifetime of HSA decreases only marginally and thus the magnitude of energy transfer efficiency is small, as obtained by time-resolved measurements. A displacement experiment predicts that the FUR binds mainly to site I but a new site having lower affinity is also observed, which shares some residues with site II as supported by molecular docking results. Results revealed that in uremia, FUR indirectly competes for Arg410, Lys414, and Ser489 with site II bound uremic toxins and directly competes for site I with site I bound uremic toxins.

Stereo-Selectivity of Human Serum Albumin to Enantiomeric and Isoelectronic Pollutants Dissected by Spectroscopy, Calorimetry and Bioinformatics

1–naphthol (1N), 2–naphthol (2N) and 8–quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (Kb) of these pollutants to HSA were moderate (104–105 M−1). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39–5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy–entropy compensation (EEC). The difference observed between ΔCp exp and ΔCp calc are suggested to be caused by binding–induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants.

A Comprehensive Insight into Binding of Hippuric Acid to Human Serum Albumin: A Study to Uncover Its Impaired Elimination through Hemodialysis

Binding of hippuric acid (HA), a uremic toxin, with human serum albumin (HSA) has been examined by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), molecular docking, circular dichroism (CD) and fluorescence spectroscopy to understand the reason that govern its impaired elimination through hemodialysis. ITC results shows that the HA binds with HSA at high (K b ∼104) and low affinity (K b ∼103) sites whereas spectroscopic results predict binding at a single site (K b∼103). The HA form complex with HSA that involves electrostatic, hydrogen and hydrophobic binding forces as illustrated by calculated thermodynamic parameters. Molecular docking and displacement studies collectively revealed that HA bound to both site I and site II; however, relatively strongly to the later. Esterase-like activity of HSA confirms the involvement of Arg410 and Tyr411 of Sudlow site II in binding of HA. CD results show slight conformational changes occurs in the protein upon ligation that may be responsible for the discrepancy in van’t Hoff and calorimetric enthalpy change. Furthermore, an increase in and is observed from DSC results that indicate increase in stability of HSA upon binding to HA. The combined results provide that HA binds to HSA and thus its elimination is hindered.

Pollutant-Induced Modulation in Conformation and β-Lactamase Activity of Human Serum Albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer’s dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aβ-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.

pH-dependent differential interacting mechanisms of sodium dodecyl sulfate with bovine serum fetuin: a biophysical insight.

Sodium dodecyl sulfate (SDS)-glycoprotein interaction serves as a model for a biological membrane. To get mechanistic insight into the interaction of SDS and glycoprotein, the effect of SDS on bovine serum fetuin (BSF) was studied in subcritical micellar concentrations at pH 7.4 and pH 2 using multiple approaches. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically via its alkyl chains at pH 7.4 up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2, as confirmed by enhanced ThT fluorescence, β-sheet formation, and TEM microscopy, whereas BSF undergoes induction of an α-helical structure in the presence of higher SDS concentration at pH 7.4. The increase in α-helical content with increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of tryptophan conformers decreases, resulting in a decrement of the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 104 M−1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (Ro) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA. Graphical abstract

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.

Biophysical insights into the interaction of hen egg white lysozyme with therapeutic dye clofazimine: modulation of activity and SDS induced aggregation of model protein.

The present study details the binding process of clofazimine to hen egg white lysozyme (HEWL) using spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and molecular docking techniques. Clofazimine binds to the protein with binding constant (Kb) in the order of 1.57 × 104 at 298 K. Binding process is spontaneous and exothermic. Molecular docking results suggested the involvement of hydrogen bonding and hydrophobic interactions in the binding process. Bacterial cell lytic activity in the presence of clofazimine increased to more than 40% of the value obtained with HEWL only. Interaction of the drug with HEWL induced ordered secondary structure in the protein and molecular compaction. Clofazimine also effectively inhibited the sodium dodecyl sulfate (SDS) induced amyloid formation in HEWL and caused disaggregation of preformed fibrils, reinforcing the notion that there is involvement of hydrophobic interactions and hydrogen bonding in the binding process of clofazimine with HEWL and clofazimine destabilizes the mature fibrils. Further, TEM images confirmed that fibrillar species were absent in the samples where amyloid induction was performed in the presence of clofazimine. As clofazimine is a drug less explored for the inhibition of fibril formation of the proteins, this study reports the inhibition of SDS-induced amyloid formation of HEWL by clofazimine, which will help in the development of clofazimine-related molecules for the treatment of amyloidosis.