Quantification of Epistemic Capacity and Physical Frailty in Chronic Kidney Disease: Koch's Disease Co-infection.

Background Chronic kidney disease (CKD) and tuberculosis (TB) co-infection devastates the affected individual physically and psychologically. Moreover, poor immune status and mental turmoil worsen cognition and quality of life. Hence, studying the cognitive function and quality of life among such patients is necessary. This study aimed to determine the changes in mini-mental state examination (MMSE) score and general health questionnaire (GHQ-12) score at six months from baseline. Methodology This prospective, observational study was conducted at Sriram Chandra Bhanja Medical College and Hospital, India, from February 2020 to December 2021. A total of 40 patients with stage 3-4 CKD and pulmonary TB were assessed with MMSE and GHQ-12 scales at baseline, two, and six months. The study population was grouped as ≤50 and >50 years of age. We used R software (version 4.1.1) for data analysis. Results In total, 40 (69%) of the 58 enrolled participants completed this study. The mean age of the study population was 50.93 ± 9.83 years. The baseline MMSE scores (≤50 years: 20.8 ± 2.1, >50 years: 20.1 ± 1.7, p = 0.17) were increased (≤50 years: 25.4 ± 1.8, >50 years: 22.4 ± 1.6, p = 0.08) at six months. The baseline GHQ-12 scores (≤50 years: 22.8 ± 2.6, >50 years: 23.1 ± 2.8, p = 0.56) were reduced (≤50 years: 17.9 ± 1.9, >50 years: 20.3 ± 2.3, p = 0.14) at six months. Conclusions The study participants' cognitive function and quality of life improved after six months of modified antitubercular drugs. Nevertheless, the intergroup differences were not statistically significant.

A novel plant lectin, NTL-125, interferes with SARS-CoV-2 interaction with hACE2.

COVID-19 caused by SARS-CoV-2 virus has had profound impact on the world in the past two years. Intense research is going on to find effective drugs to combat the disease. Over the past year several vaccines were approved for immunization. But SARS-CoV-2 being an RNA virus is continuously mutating to generate new variants, some of which develop features of immune escape. This raised serious doubts over the long-term efficacy of the vaccines. We have identified a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, which effectively inhibits SARS-CoV-2 replication in Vero-E6 cell line. In silico docking studies revealed that NTL-125 has strong affinity to viral Spike RBD protein, preventing it from attaching to hACE2 receptor, the gateway to cellular entry. Binding analyses revealed that all the mutant variants of Spike protein also have stronger affinity for NTL-125 than hACE2. The unique α-helical tail of NTL-125 plays most important role in binding to RBD of Spike. NTL-125 also interacts effectively with some glycan moieties of S-protein in addition to amino acid residues adding to the binding strength. Thus, NTL-125 is a highly potential antiviral compound of natural origin against SARS-CoV-2 and may serve as an important therapeutic for management of COVID-19.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India.

BACKGROUND: Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. METHODS: This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450-680 was also carried out to identify the polymorphism. RESULTS: A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. CONCLUSIONS: The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Real-Time Observation of Single Atoms Trapped and Interfaced to a Nanofiber Cavity.

We demonstrate efficient interfacing of individually trapped single atoms to a nanofiber cavity. The cavity is formed by fabricating photonic crystal structures directly on the nanofiber using femtosecond laser ablation. The single atoms are interfaced to the nanofiber cavity using an optical tweezer based side-illumination trapping scheme. We show that the fluorescence of individual single atoms trapped on the nanofiber cavity can be readily observed in real-time through the fiber guided modes. From the photon statistics measured for different cavity decay rates, the effective coupling rate of the atom-cavity interface is estimated to be 34±2 MHz. This yields a cooperativity of 5.4±0.6 (Purcell factor=6.4±0.6) and a cavity enhanced channeling efficiency as high as 85±2% for a cavity mode with a finesse of 140. The trap lifetime is measured to be 52±5 ms. These results may open new possibilities for deterministic preparation of single atom events for quantum photonics applications on an all-fiber platform.

Photothermal tuning and stabilization of a photonic crystal nanofiber cavity.

We report the photothermal properties of a photonic crystal nanofiber (PhCN) cavity, which is fabricated using a femtosecond laser ablation technique, under ultra-high vacuum conditions. The results show that by launching a non-resonant guided light, the stopband of the PhCN, along with the cavity modes, can be tuned at a rate of 250 GHz (∼0.5 nm)/mW. Moreover, due to the thermal self-locking effect of a resonant light, a cavity mode with a finesse of 25 can be tuned over a few free spectral ranges using only a few µW of guided light. As an enabling step, we demonstrate a dual-mode locking scheme to thermally stabilize a cavity mode to the atomic line for single atom-based cavity quantum electrodynamics experiments.

Coherent interaction of orthogonal polarization modes in a photonic crystal nanofiber cavity.

We show that coherent interaction between two sets of multiple resonances leads to exotic resonant effects, such as Fano-type resonances, optical analogue of electro-magnetically induced transparency, and avoided crossing between modes, under different coupling regimes. We experimentally demonstrate such resonant effects in a photonic crystal nanofiber cavity using two sets of cavity modes with orthogonal polarizations. The interaction between the modes arises due to intra-cavity polarization mixing. The observed line shapes are reproduced using a multiple-mode interaction model. Such spectral characteristics may further enhance the capabilities of the nanofiber cavity as a fiber-in-line platform for nanophotonics and quantum photonics applications.

Microzonation, ecological risk and attributes of metals in highway road dust traversing through the Kaziranga National Park, Northeast India: implication for confining metal pollution in the national forest.

Despite the abundant literature on metal contamination through road dust (RD) in urban/suburban and residential/highway regions, the RD of highways traversing through the Kaziranga National Park, NE India, has not been studied and lacks baseline data. The objective of the present study was to ascertain the possibility of highway microzonation based on temporal and spatial variability of metal pollution level and ecological risk. It was found that the RD contains an average content of (1.7-33.5 mg/kg) for Cd, Co, Cu and Pb and (121-574 mg/kg) for Ni, Zn, Cr and Mn across the highway passing through the national forest attributed by various sources. The study revealed three possible microzones present in the studied highway NH-37 based on spatial trend of metal as well as human interference. An attempt was made to understand the possible source of metals by principal component analysis, and four sources were identified: Three were of vehicular origin, and another was related to road surface and subsurface condition. The use of noise barrier walls as an effective measure to control the translocation of RD from one place to other was recommended to reduce the hostile effects of metal accumulation in the sensitive ecosystem. Thus, the study suggested and necessitated micronizing the system based on human interference level, ecological risk factors, spatial variability of pollutants and traffic pattern for their efficient management and conservation.

Inhibition of Insulin Amyloid Fibrillation by a Novel Amphipathic Heptapeptide: MECHANISTIC DETAILS STUDIED BY SPECTROSCOPY IN COMBINATION WITH MICROSCOPY.

The aggregation of insulin into amyloid fibers has been a limiting factor in the development of fast acting insulin analogues, creating a demand for excipients that limit aggregation. Despite the potential demand, inhibitors specifically targeting insulin have been few in number. Here we report a non-toxic and serum stable-designed heptapeptide, KR7 (KPWWPRR-NH2), that differs significantly from the primarily hydrophobic sequences that have been previously used to interfere with insulin amyloid fibrillation. Thioflavin T fluorescence assays, circular dichroism spectroscopy, and one-dimensional proton NMR experiments suggest KR7 primarily targets the fiber elongation step with little effect on the early oligomerization steps in the lag time period. From confocal fluorescence and atomic force microscopy experiments, the net result appears to be the arrest of aggregation in an early, non-fibrillar aggregation stage. This mechanism is noticeably different from previous peptide-based inhibitors, which have primarily shifted the lag time with little effect on later stages of aggregation. As insulin is an important model system for understanding protein aggregation, the new peptide may be an important tool for understanding peptide-based inhibition of amyloid formation.

Cavity quantum electrodynamics on a nanofiber using a composite photonic crystal cavity.

We demonstrate cavity QED conditions in the Purcell regime for single quantum emitters on the surface of an optical nanofiber. The cavity is formed by combining an optical nanofiber and a nanofabricated grating to create a composite photonic crystal cavity. By using this technique, significant enhancement of the spontaneous emission rate into the nanofiber guided modes is observed for single quantum dots. Our results pave the way for enhanced on-fiber light-matter interfaces with clear applications to quantum networks.

Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Photonic crystal nanofiber using an external grating.

We implemented a photonic crystal nanofiber device by reversibly combining an optical nanofiber and a nanofabricated grating. Using the finite-difference time-domain method, we designed the system for minimal optical loss while tailoring the resonant wavelength and bandwidth of the device. Experimentally, we demonstrated that the combined system shows a strong photonic stop band in good agreement with numerical predictions. The resulting device may be used to realize strong light-matter coupling near the nanofiber surface.

Novel role of Wag31 in protection of mycobacteria under oxidative stress.

Wag31 of Mycobacterium tuberculosis belongs to the DivIVA family of proteins known to regulate cell morphology in Gram-positive bacteria. Here we demonstrate an unrecognized, novel role of Wag31 in oxidatively stressed mycobacteria. We report the cleavage of penicillin-binding protein 3 (PBP3) by the intramembrane metalloprotease Rv2869c (MSMEG_2579) in oxidatively stressed cells. Amino acids (102)A and (103)A of PBP3 are required for Rv2869c-mediated cleavage. Wag31(MTB), by virtue of its interaction with PBP3 through amino acid residues (46)NSD(48), protects it from oxidative stress-induced cleavage. PBP3 undergoes cleavage in Mycobacterium smegmatis (strain PM2) harbouring wag31(Delta(46)NSD(48)) instead of the wild type, with concomitant reduction in ability to withstand oxidative stress. Overexpression of Wag31(Delta(46)NSD(48)) attenuates the survival of M. tuberculosis in macrophages with concomitant cleavage of PBP3, and renders the organism more susceptible towards hydrogen peroxide as well as drugs which generate reactive oxygen species, namely isoniazid and ofloxacin. We propose that targeting Wag31 could enhance the activity of mycobactericidal drugs which are known to generate reactive oxygen species.

Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin.

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.

Unfolding studies of Escherichia coli maltodextrin glucosidase monitored by fluorescence spectroscopy.

Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.

Chaperone-mediated inhibition of tubulin self-assembly.

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.

Purification and characterization of alpha-amylase from Bacillus amyloliquefaciens NCIM 2829.

Alpha-Amylase (EC 3.2.1.1) was purified to homogeneity (specific activity 58,000 micromole min(-1) mg protein(-1)) from the culture filtrate of Bacillus amyloliquefaciens NCIM 2829. Its molecular mass was found to be 67.5 kDa. The activity of the enzyme increased by almost 50% in the presence of Co+2 ion. Hg2+ and Cu2+ acted as strong inhibitors of the enzyme. The tryptophan moities of the enzyme were fairly protected from the aqueous environment. However, the globular interior of the protein was somewhat loosely packed. The protein had nearly an equal amount of alpha-helical and beta-sheet structure in dilute solution. In concentrated solution, its secondary structure had a higher proportion of beta-sheet at the expense of some random coil structure. The protein showed a molten globule state at a low concentration of chaotropic agent. The denaturation profile of the protein showed no cooperativity. Co2+ enhanced the structural stability of the enzyme.

Role of ATP on the interaction of alpha-crystallin with its substrates and its implications for the molecular chaperone function.

ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone alpha-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of alpha-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on alpha-crystallin, but no appreciable enhancement of the same was observed for the substrate protein gamma-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mgm concentration stabilizes the alpha-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone alpha-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to alpha-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5'-O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of alpha-crystallin in vivo is discussed.

MAP2 prevents protein aggregation and facilitates reactivation of unfolded enzymes.

It is well established that in addition to its functional role in cell motility, cell division and intracellular transport, cytoskeletal protein tubulin also possesses significant chaperone-like activity. In vitro studies from our laboratory showed that dimeric tubulin can prevent stress induced aggregation of substrate proteins, can resist thermal deactivation of enzymes and can also refold enzymes from their fully denatured state [Manna, T., Sarkar, T., Poddar, A., Roychowdhury, M., Das, K.P. & Bhattacharyya, B. (2001) J. Biol. Chem.276, 39742-39747]. Negative charges of the C-termini of both subunits of tubulin are essential for this chaperone-like property as the deletion of only beta-C-terminus or the binding of a 14-residue basic peptide P2 to the alpha-C-terminus completely abolishes this property [Sarkar, T., Manna, T., Bhattacharyya, S., Mahapatra, P., Poddar, A., Roy, S., Pena, J., Solana, R., Tarazona, R. & Bhattacharyya, B. (2001) Proteins Struct. Funct. Genet.44, 262-269]. Based on these results, one would expect that the microtubular proteins (MTP, tubulin with microtubular-associated proteins, i.e. MAPs bound to the C-terminus) should not possess any chaperone-like activity. To our surprise we noticed excellent chaperone-like activity of MTP. MTP prevents chemical and thermal aggregation of other proteins and can enhance the extent of refolding of fully unfolded substrate enzymes. Because MTP contains tubulin as well as several MAPs bound to the C-termini of tubulin, we fractionated and purified microtubular associated protein 2 (MAP2) and tau using phosphocellulose chromatography. Experiments with purified proteins demonstrated that it is the MAP2 of MTP that exhibits significant chaperone-like activity. This has been shown by the prevention of dithiothreitol-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase and regain of enzymatic activity during refolding of unfolded substrates. Tau, which shares a homologous C-terminal domain with MAP2, possesses no such activity.

SDS induced structural changes in alpha-crystallin and it's effect on refolding.

Alpha-crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. Alpha-crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the beta-sheet structure was diminished followed by concomitant increase in the alpha-helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric gamma-crystallin, which is also a major beta-sheet protein like alpha-crystallin. When alpha-crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of alpha-helix was more than that incubated directly in the same concentration of SDS. The results show that alpha-crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.

BisANS binding to tubulin: isothermal titration calorimetry and the site-specific proteolysis reveal the GTP-induced structural stability of tubulin.

Interactions of bisANS and ANS to tubulin in the presence and absence of GTP were investigated, and the binding and thermodynamic parameters were determined using isothermal titration calorimetry. Like bisANS binding to tubulin, we observed a large number of lower affinity ANS binding sites (N1 = 1.3, K1 = 3.7 x 10(5) M(-1), N2 = 10.5, K2 = 7 x 10(4)/M(-1)) in addition to 1-2 higher affinity sites. Although the presence of GTP lowers the bisANS binding to both higher and lower affinity sites (N1 = 4.3, N2 = 11.7 in absence and N1 = 1.8, N2 = 3.6 in presence of GTP), the stoichiometries of both higher and lower affinity sites of ANS remain unaffected in the presence of GTP. BisANS-induced structural changes on tubulin were studied using site-specific proteolysis with trypsin and chymotrypsin. Digestion of both alpha and beta tubulin with trypsin and chymotrypsin, respectively, has been found to be very specific in presence of GTP. GTP has dramatic effects on lowering the extent of nonspecific digestion of beta tubulin with trypsin and stabilizing the intermediate bands produced from both alpha and beta. BisANS-treated tubulin is more susceptible to both trypsin and chymotrypsin digestion. At higher bisANS concentration (>20 microM) both alpha and beta tubulins are almost totally digested with enzymes, indicating bisANS-induced unfolding or destabilization of tubulin structure. Again, the addition of GTP has remarkable effect on lowering the bisANS-induced enhanced digestion of tubulin as well as stabilizing effect on intermediate bands. These results of isothermal titration calorimetry, proteolysis and the DTNB-kinetics data clearly established that the addition of GTP makes tubulin compact and rigid and hence the GTP-induced stabilization of tubulin structure. No such destabilization of tubulin structure has been noticed with ANS, although, like bisANS, ANS possesses a large number of lower affinity binding sites. On the basis of these results, we propose that the unique structure of bisANS, which in absence of GTP can bind tubulin as a bifunctional ligand (through its two ANS moieties), is responsible for the structural changes of tubulin.