Basir Ahmad - Academia.edu (original) (raw)

Papers by Basir Ahmad

Hesperidin, a flavanone glycoside shows high antioxidant properties and posses ability to go thro... more Hesperidin, a flavanone glycoside shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s and systemic amyloidoses. In this work, we investigated the potential of hesperidin (HESP) to interact with hen egg white lysozyme (HEWL) monomer and prevent its aggregation. The HESP-HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (Ka, ~ 5104 M-1) mainly through hydrogen bonding, water bridges and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulphide reducing agent tries-(2-carboxyethyl) phosphine (TCEP). Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.

The molecule, 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol (BBE) is known to act as a selective inhib... more The molecule, 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol (BBE) is known to act as a selective inhibitor of poliovirus, rhinovirus, Candida albicans, several bacterial species, and is easily synthesized by Phillips reaction. The interaction of BBE with BSA and the effects of its binding on the conformation and unfolding/refolding pathways of the protein were investigated using multispectroscopic techniques and molecular modeling. The binding studies indicate that BSA has one high affinity BBE binding site with association constant 6.02±0.05×10(4) M(-1) at 298 K. By measuring binding at different temperatures, we determined the changes in enthalpy (ΔH = -15.13±2.15 kJ mol(-1)), entropy (ΔS = 40.87±7.25 J mol(-1) K(-1)) and free energy (ΔG( = )26.78±1.02) of interaction, which indicate that the binding was spontaneous and both enthalpically and entropically driven. Based on molecular modeling and thermodynamic parameters, we proposed that the complex formation involved mainly hydrophilic interaction such as hydrogen bonding between hydroxyl groups of ethane-1,2-diol fragment with Tyr410 and benzimidazole sp(2) nitrogen atom with Ser488 and hydrophobic interaction between phenyl ring of one benzimidazole of the ligand and hydrophobic residues namely, Ile387, Cys391, Phe402, Val432 and Cys437. The sequential unfolding mechanism of BSA, site-specific marker displacement experiments and molecular modeling showed that the molecule preferably binds in subdomain IIIA. The BBE binding to BSA was found to cause both secondary and tertiary structural alterations in the protein as studied by intrinsic fluorescence, near-UV and far-UV circular dichroism results. The unfolding/refolding study showed that BBE stabilized native to intermediate states (N⇌I) transition of the protein by ∼2 kJ mol(-1) without affecting the intermediate to unfolded states (I⇌U) transition and general mechanism of unfolding of BSA.

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, bu... more Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

Biophysical Journal, 2011

It is generally accepted that aggregation takes place from partially unstructured states of initi... more It is generally accepted that aggregation takes place from partially unstructured states of initially globular proteins or unstructured states of intrinsically disordered peptides or proteins. However, we know very little about the elementary steps of protein aggregation. The intramolecular diffusion due to formation of contact between two points in a polypeptide chain may be the earliest step of the folding and also aggregation. It is not yet known how intramolecular diffusion of a polypeptide chain is affected under aggregating conditions. In particular it is unknown whether the rate of intramolecular diffusion under physiological conditions is decreased/increased or completely vanished under aggregating conditions. We have addressed this issue by studying the intramolecular contacts formation in an intrinsically disordered protein, a-synuclein under both physiological and aggregating conditions. At low temperatures, the rate of intramolecular diffusion is quite high, comparable to unstructured model peptides. However this rate decreases with increasing temperature, suggesting that the protein reconfigures more slowly under aggregating conditions. Parkinson's disease (PD) is the second most common neurodegenerative disease, with the number one risk factor being age. Despite its prevalence and extensive research efforts at many levels to understand the molecular disease mechanism and identify effective drug targets (of which there are currently none), much remains to be understood about PD, including the role of the heavily implicated protein a-synuclein. This small and intrinsically disordered protein (IDP) is the primary component of Lewy bodies (intraneuronal inclusions pathologically associated with PD) and has been linked genetically to PD through mutation and overexpression-linked toxicity. Structural investigation by conventional bulk methods has proven to be quite challenging due to the inherent flexibility of the molecule and subsequent heterogeneity of the population. To reveal critical information about the ''misfolding'' (or perhaps simply alternative folding) behavior of a-synuclein, especially as it relates to aggregation, single-molecule Förster Resonance Energy Transfer (smFRET) techniques were applied to wild-type and disease-related variants of a-synuclein. The monomeric protein was then observed in misfolding-related conditions, revealing distinct folding characteristics of aggregation-related conformations, which may eventually lead to new insights into a-synuclein's primary role in human biology by comparison.

PLOS One, 2012

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol ... more 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 HSAhydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for b-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 b-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 b-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

Biochemistry-moscow, 2010

We have studied the effect of trifluoroethanol (TFE) on the native (pH 7.0), acid unfolded (pH 2.... more We have studied the effect of trifluoroethanol (TFE) on the native (pH 7.0), acid unfolded (pH 2.6), and molten globule (pH 1.4) states of glucose oxidase (GOX) by circular dichroism and fluorescence spectroscopy. In the presence of 50% TFE, at pH 7.0 and 2.6, GOX exhibited a transition from native coiled-coil and acid unfolded state to non-associating α-helical state. Interestingly, at pH 1.4, 15% TFE induced the formation of β-structured intermediate by loss of 1-anilino-8-naphthalenesulfonate binding site and almost all tertiary contacts. The β-structured intermediate converted into open helical conformation on further addition of TFE.

Protein and Peptide Letters, 2009

Proteins may form undesirable aggregates during the process of folding. Increasing evidence sugge... more Proteins may form undesirable aggregates during the process of folding. Increasing evidence suggests that amyloid fibrils may arise from partially folded precursor molecules. We have previously demonstrated that hen egg white lysozyme [HEWL] exists as molten globule at pH 12.7. Here, we report that lysozyme at pH 7.0 and 11.0 are nearly stable to the addition of up to 45% t-butanol, but treatment of the alkali-induced molten globule form of HEWL [AMGL] with 20% t-butanol caused the formation of amyloid-like fibrils as evidenced by enhanced Thioflavin T binding and DLS measurements.

Protein and Peptide Letters, 2010

The conformation of bovine serum fetuin (BSF) was examined over the pH 7.0-12.9 regions by circul... more The conformation of bovine serum fetuin (BSF) was examined over the pH 7.0-12.9 regions by circular dichroism, intrinsic fluorescence and ANS binding. We observed that at higher pH, BSF exists in alkaline unfolded state. Our results provided evidence that correlates simultaneous formation of secondary structure followed by accumulation of hydrophobic clusters.

Biochemistry-moscow, 2009

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a fu... more Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.

Amino Acids, 2010

Keyhole limpet hemocyanin (KLH) is widely used as an immune stimulant and hapten carrier derived ... more Keyhole limpet hemocyanin (KLH) is widely used as an immune stimulant and hapten carrier derived from a marine mollusc Megathura crenulata. To provide details of the stability and equilibrium of KLH, different intermediate species were investigated with a series of biophysical techniques: circular dichroism, binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid, acrylamide-induced fluorescence quenching, thermal stability and dynamic light scattering. KLH in its native state at pH 7.4 exists in the stable didecameric form with hydrodynamic radii (R h) of 28.22 nm, which is approximately equal to a molecular mass of 8.8 ± 0.6 MDa. The experimental results demonstrated the presence of two structurally distinct species in the conformational transition of KLH under acidic conditions. One species populates at pH 2.8, characterized as decameric (4.8 ± 0.2 MDa; R h = 22.02 nm), molten globule-like state, while the other accumulates at pH 1.2 and is characterized as a tetramer (2.4 ± 0.8 MDa; R h = 16.47 nm) with more organized secondary and tertiary structures. Our experimental manipulation of the oligomeric states of KLH has provided data that correlate well with the known oligomeric forms obtained from total KLH formed in vivo and extends our understanding of multimer formation by KLH. The results are of particular interest in light of the important role of the mechanistic pathway of pH-dependent structural changes of Hc stability in the biochemical and medical applications of these respiratory proteins.

Biocatalysis and Biotransformation, 2007

ABSTRACT Cross-linking of the protease stem bromelain (bromelain) with 0.25 and 1.25% glutaraldeh... more ABSTRACT Cross-linking of the protease stem bromelain (bromelain) with 0.25 and 1.25% glutaraldehyde (GTA) results in the formation of a large molecular mass, multimeric and soluble aggregate having comparable activity to the unmodified bromelain. Both 0.25 and 1.25% GTA cross-linked (CL) bromelain preparations were more stable against urea, guanidine hydrochloride (GdnHCl) and temperature-induced inactivation, and exhibited slightly better storage stability compared to the unmodified protease. Such a high molecular weight, soluble, active and stable preparation may be useful in industry, i.e. in the textile industry for improving the properties of a fabric without loss of fabric strength and shape.

Journal of Biological Chemistry, Jan 1, 2012

Background: ␣-Synuclein is an aggregation-prone protein that reconfigures more slowly under aggre... more Background: ␣-Synuclein is an aggregation-prone protein that reconfigures more slowly under aggregating conditions. Results: Curcumin binds to monomeric ␣-synuclein, prevents aggregation, and increases the reconfiguration rate, particularly at high temperatures. Conclusion: Curcumin rescues the protein from aggregation by making the protein more diffusive. Significance: The search for aggregation inhibitors should account for changes in chain dynamics by the small molecule. Downloaded from FIGURE 5. a and b, reaction-limited and diffusion-limited rates, respectively, for mutant A69C/F94W in the absence and presence of various concentrations of curcumin. The diffusion-limited rates were calculated for the viscosity of buffer at each temperature. c, average Trp-Cys distance calculated using Equations 3 and 5. d, diffusion coefficients calculated from measured k Dϩ and Gaussian probability distributions using Equation 4.

We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is... more We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is controlled by the rate of backbone reconfiguration. When reconfiguration is fast, bimolecular association is not stable, but as reconfiguration slows, association is more stable and subsequent aggregation is faster. To investigate this hypothesis, we have measured the rate of intramolecular diffusion in α-synuclein, a protein involved in Parkinson's disease, under solvent conditions that accelerate or decelerate aggregation. Using the method of tryptophan-cysteine (Trp-Cys) quenching, the rate of intramolecular contact is measured in four different loops along the chain length. This intrinsically disordered protein is highly diffusive at low temperature at neutral pH, when aggregation is slow, and compacts and diffuses more slowly at high temperature or low pH, when aggregation is rapid. Diffusion also slows with the disease mutation A30P. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.

Biochimica et Biophysica Acta ( …, Jan 1, 2010

International journal of biological …, Jan 1, 2010

Archives of biochemistry and …, Jan 1, 2006

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2... more Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable a-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both b-sheet and a-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from b-sheet to a-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.

IUBMB life, Jan 1, 2007

Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by ... more Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state *pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase.

Journal of …, Jan 1, 2007

Some hemipteran xylem and phloem-feeding insects have evolved specialized alimentary structures o... more Some hemipteran xylem and phloem-feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber is likely facilitated by an aquaporin protein. B. tabaci aquaporin-1 (BtAQP1) possesses characteristic aquaporin topology and conserved pore-forming residues found in water-specific aquaporins. As predicted for an integral transmembrane protein, recombinant BtAQP1 expressed in cultured insect cells localized within the plasma membrane. BtAQP1 is primarily expressed in early instar nymphs and adults, where in adults it is localized in the filter chamber and hindgut. Xenopus oocytes expressing BtAQP1 were water permeable and mercury-sensitive, both characteristics of classical water-specific aquaporins. These data support the hypothesis that BtAQP1 is a water transport protein within the specialized filter chamber of the alimentary tract and functions to translocate water across tissues for maintenance of osmotic pressure and/or excretion of excess dietary fluid.

Journal of biochemistry, Jan 1, 2006

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the ... more We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.

International journal of biological …, Jan 1, 2008

The user has requested enhancement of the downloaded file. All in-text references underlined in b... more The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.