Free spermidine evokes superoxide radicals that manifest toxicity.

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Thus, wild-type E. coli can tolerate applied exogenous spermidine stress, but ΔspeG strain of E. coli fails to do that. Here, using different reactive oxygen species (ROS) probes and performing electron paramagnetic resonance spectroscopy, we provide evidence that although spermidine mitigates oxidative stress by lowering overall ROS levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the ΔspeG strain. Furthermore, performing microarray experiment and other biochemical assays, we show that the spermidine-induced superoxide anions affected redox balance and iron homeostasis. Finally, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Therefore, we propose that the spermidine-induced superoxide generation is one of the major causes of spermidine toxicity in E. coli.

Emerging Role of Mass Spectrometry-Based Structural Proteomics in Elucidating Intrinsic Disorder in Proteins.

Inherent disorder is an integral part of all proteomes, represented as fully or partially unfolded proteins. The lack of order in intrinsically disordered proteins (IDPs) results in an incredibly flexible, floppy, and heterogeneous ensemble, contrary to the well-structured and unique organization of folded proteins. Despite such unusual demeanor, IDPs are crucial for numerous cellular processes and are increasingly being associated with disease-causing pathologies. These warrant more intensive investigation of this atypical class of protein. Traditional biophysical tools, however, fall short of analyzing IDPs, thus making their structure-function characterization challenging. Mass spectrometry (MS) in recent years has evolved as a valuable tool for elucidating the unusual conformational facets of IDPs. In this review, the features of advanced MS techniques such as Hydrogen-deuterium exchange (HDX)-MS, native MS, limited proteolysis (LiP)-MS, chemical cross-linking (XL)-MS, and Fast photochemical oxidation of proteins (FPOP)-MS are briefly discussed. Recent MS studies on IDPs and the unique advantages/shortfalls associated with the above methods while evaluating structural proteomics of IDPs, are illustrated. Eventually the future scope of the MS methods in further decoding the unexplored landscapes of IDPs is presented.

Application of native mass spectrometry in studying intrinsically disordered proteins: A special focus on neurodegenerative diseases.

Intrinsically disordered proteins (IDPs) are integral part of the proteome, regulating vital biological processes. Such proteins gained further visibility due to their key role in neurodegenerative diseases and cancer. IDPs however, escape structural characterization by traditional biophysical tools owing to their extreme flexibility and heterogeneity. In this review, we discuss the advantages of native mass spectrometry (MS) in analysing the atypical conformational dynamics of IDPs and recent advances made in the field. Especially, MS studies unravelling the conformational facets of IDPs involved in neurodegenerative diseases are highlighted. The limitations and the future promises of native MS while studying IDPs have been discussed.

Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer.

BACKGROUND: Aggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer. METHODS: We synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching. RESULTS: Deuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule. CONCLUSIONS: PHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high ß-structure propensity in second and third MTBR (R2, R3), thus preventing intermolecular interaction between them, or by imparted rigidity in R2-R3, preventing the formation of extended ß-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly. GENERAL SIGNIFICANCE: Our findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.

Conformational heterogeneity of tau: Implication on intrinsic disorder, acid stability and fibrillation in Alzheimer's disease.

The self-assembly of intrinsically disordered protein tau into paired helical filament forms one of the hallmarks of Alzheimer's disease. However, the facets of innately disordered structure of tau and its conversion to a ß-sheet-rich fibril during several tauopathies are poorly understood. Here, we provide a direct insight into the ensemble of highly heterogeneous conformational families of tau at physiological pH, by nano-electrospray mass spectrometry coupled with ion mobility. The average collision cross section of the most unfolded conformer was higher by >2 fold than that of the most folded one. Acidic pH largely induced unfolding in tau, obliterating the compact conformers completely. The highly unfolded conformers were the key species bestowing the unusual solubility to tau at low pH, with limited contribution from intramolecular long-range interfaces giving rise to ordered conformers. Contrarily, alkaline pH shifted tau towards folded conformations due to charge neutralization, keeping the overall random coil architecture intact. Intriguingly, the heparin-induced in vitro aggregation propensity of the protein attenuated at both acidic and alkaline pH, illustrating the significance of altered conformations in pathological functions of tau. Our observations at low pH indicate that a reorganization of the intricate network of momentary long-range contacts in tau might have implication in its aggregation pathology. Disease-modifying therapies for Alzheimer's disease targeting either to disrupt the essential fibril-forming interaction at third microtubule-binding repeat of tau or to perturb specific binding interaction of tau with endogenous polyanionic species might be of high benefit.

Molecular insights of inhibition in sickle hemoglobin polymerization upon glutathionylation: hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulation-based approach.

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of ßVal6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of ßCys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of ßVal6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia.

Effect of altered solution conditions on tau conformational dynamics: Plausible implication on order propensity and aggregation.

Intrinsically disordered protein tau plays a central role in maintaining neuronal network by stabilizing microtubules in axon. Tau reportedly possesses random coil architecture, which is largely inert to alteration in solution conditions. However, the presence of transient compact conformers and residual structure has been evident from previous reports. Also, during Alzheimer's disease, misfolded tau detaches from microtubule and forms ordered filaments, which is the hallmark of the disease. Despite its fundamental role in neuronal physiology and in pathological cascade of several fatal neurodegenerative diseases, tau conformational dynamics remains poorly understood. In the present study, we have explored the effect of ionic strength, temperature and solvent polarity on tau40 conformational preferences using ion mobility mass spectrometry. Investigation of collision cross section revealed that while low ionic strength, elevated temperature and reduced solvent polarity mostly induced partial collapse in tau40 conformers, higher ionic strength led to an expansion of the molecule. Limited proteolysis identified segments of tau40 projection domain and proline-rich region having high order propensity and a C-terminal region having vulnerability for further expansion at altered solution conditions. The high susceptibility for disorder-to-order transition in the above region of the protein might have crucial implication on its role as microtubule spacers, and in cellular signaling cascade. The conformational adaptation of tau40 did not enhance the heparin-induced aggregation proclivity of the protein. Nevertheless, the observed correlation of electrostatic interaction with fibrillation propensity of tau40 might indicate plausible link between hyperphosphorylation at diseased state with tau conformation and self-assembly.

Adsorption Induced Changes of Human Hemoglobin on Ferric Pyrophosphate Nanoparticle Surface Probed by Isotope Exchange Mass Spectrometry: An Implication on Structure-Function Correlation.

In general, proteins in the biological system interact with nanoparticles (NPs) via adsorption on the particle surface. Understanding the adsorption at the molecular level is crucial to explore NP-protein interactions. The increasing concerns about the risk to human health on NP exposure have been explored through the discovery of a handful protein biomarkers and biochemical analysis. However, detailed information on structural perturbation and associated functional changes of proteins on interaction with NPs is limited. Erythrocytes (red blood cells) are devoid of defense mechanism of protecting NP penetration through endocytosis. Therefore, it is important to investigate the interaction of erythrocyte proteins with NPs. Hemoglobin, the most abundant protein of human erythrocyte, is a tetrameric molecule consisting of α- and ß-globin chains in duplicate. In the present study, we have used hemoglobin as a model system to investigate NP-protein interaction with ferric pyrophosphate NPs [NP-Fe4(P2O7)3]. We report the formation of a bioconjugate of hemoglobin upon adsorption to NP-Fe4(P2O7)3 surface. Analysis of the bioconjugate indicated that Fe3+ ion of NP-Fe4(P2O7)3 contributed in the bioconjugate formation. Using hydrogen/deuterium exchange based mass spectrometry, it was observed that the amino termini of α- and ß-globin chains of hemoglobin were involved in the adsorption on NP surface whereas the carboxy termini of both chains became more flexible in its conformation compared to the respective regions of the normal hemoglobin. Circular dichroism spectra of desorbed hemoglobin indicated an adsorption induced localized structural change in the protein molecule. The formation of bioconjugate led to functional alteration of hemoglobin, as probed by oxygen binding assay. Thus, we hypothesize that the large amount of energy released upon adsorption of hemoglobin to NP surface might be the fundamental cause of structural perturbation of human hemoglobin and subsequent formation of the bioconjugate with an altered function.

Mass Spectrometry-Based Diagnosis of Hemoglobinopathies: A Potential Tool for the Screening of Genetic Disorder.

Hemoglobinopathies are caused by point mutation in globin gene that results in structural variant of hemoglobin. While 7 % of world populations are carrier of hemoglobinopathies, the prevalence of the disease varies between 3 to 17 % across different population groups in India. In a diagnostic laboratory, alkaline gel electrophoresis and cation exchange-based HPLC (CE-HPLC) are most widely used techniques for characterization of hemoglobin variants. In the above methods, the differential surface charge of hemoglobin molecule in variants is exploited for their characterization. Sometime, co-migration of variants in gel electrophoresis and co-elution or elution with unknown retention time in automated CE-HPLC might lead to ambiguity in the analysis of hemoglobinopathies. Under such circumstances, it is necessary to use other analytical methods that provide unambiguous results. Mass spectrometry-based proteomics approach and DNA sequence analysis are examples of such alternative methods. In the present study, liquid chromatography coupled to mass spectrometry has been used for three commonly observed variants in India, e.g., HbE, HbQ India and HbD Punjab that appeared with inappropriate results in the conventional analysis. A customized hemoglobin variant database has been used in the mass spectrometry-based analysis of those three variants. Mass spectrometry-based proteomics approach was used to analyze above variant sample accurately.

Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.

To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explore the conformational transition associated with oxygenation of hemoglobin in vivo, the isotope exchange kinetics was simplified using the method of initial rates. RBC might be considered as an in vivo system of pure hemoglobin. Thus, as a proof-of-concept, the observed results were correlated with structural transition of hemoglobin associated with its function established in vitro. This is the first report on structural changes of a protein upon ligand binding in its endogenous environment. The proposed method might be applicable to proteins in their native state, irrespective of location, concentration, and size. The present in-cell approach opens a new avenue to unravel a plethora of biological processes like ligand binding, folding, and post-translational modification of proteins in living cells.

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample.

Glycated hemoglobin (HbA1c) is a 'gold standard' biomarker for assessing the glycemic index of an individual. HbA1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the ß-globin chain. Cation exchange based high performance liquid chromatography (CE-HPLC) is mostly used to quantify HbA1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE-HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA1c (52.1%) in the CE-HPLC method. The observed HbA1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA1c and Hb Beckman might have resulted in the coelution of the variant with HbA1c in CE-HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA1c must be estimated using an alternative method.

MAP2c prevents arachidonic acid-induced fibril formation of tau: Role of chaperone activity and phosphorylation.

Tau has long been associated with Alzheimer's disease, where it forms neurofibrillary tangles. Here we show for the first time by electron microscopy that MAP2c prevents arachidonic acid-induced in vitro aggregation of tau. However, phosphorylated MAP2c failed to prevent the same. Previously we reported that MAP2c possesses chaperone-like activity while tau does not (Sarkar et al., 2004, Eur J Biochem., 271(8), 1488-96). Here we demonstrate that phosphorylation severely impaired the chaperone activity of MAP2c, implying a crucial role of chaperone in preventing tau fibrillation. Additionally, the ability of MAP2c to induce microtubule polymerization was abolished completely upon phosphorylation. As tau and MAP2c possess highly homologous C-termini, we speculated that the N-terminus of MAP2c might account for its chaperone activity. Nevertheless, experiments showed that N-terminus of MAP2c alone is inactive as a chaperone. Our preliminary findings suggest that MAP2c/MAP2 could be one of the regulators maintaining tau homeostasis in the cell.

Automated analysis of hemoglobin variants using nanoLC-MS and customized databases.

Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC-MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC-MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis.

Glutathionylation induced structural changes in oxy human hemoglobin analyzed by backbone amide hydrogen/deuterium exchange and MALDI-mass spectrometry.

Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and ß globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: ß86-102, ß1-14, α34-46, ß32-41, ß130-146, ß115-129, ß73-81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.

Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium exchange and MALDI mass spectrometry.

Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: α34-46, α1-29, ß32-41, ß86-102, ß115-129, and ß130-146. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.

A femtosecond study of the interaction of human serum albumin with a surfactant (SDS).

The interaction of a protein, human serum albumin (HSA) with a surfactant (sodium dodecyl sulfate, SDS) was studied by femtosecond up-conversion. HSA was labeled covalently with a probe (CPM, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin). Binding of SDS to HSA is found to accelerate the solvation dynamics approximately 1.3-fold. The solvation dynamics in HSA displays two time components: 30 ps (20 %) and 800 ps (80 %). When approximately 10 SDS molecules bind to HSA the components are 15 ps (40 %) and 800 ps (60 %). It is argued that SDS may increase the solvent exposure of the probe (CPM); it may also displace the buried water molecules in the immediate vicinity of CPM.

Modulation of digestive enzyme activities during ontogeny of Labeo rohita larvae fed ascorbic acid enriched zooplankton.

The effect of supplementation of ascorbic acid through enriched zooplankton [10%, 20% and 30% ascorbyl palmitate (AP) inclusion in diet of zooplankton] on different digestive enzyme activities during ontogeny of Labeo rohita larvae was studied from 4 day to 15 day post hatch. Ascorbic acid (AA) content in different groups of unenriched (8.6+/-0.71) and enriched zooplankton were, 750+/-29.3, 1409.1+/-45.5, 2009.21+/-199.2 mug/g respectively on dry matter basis with differences (P<0.05) between the treatments. A difference (P<0.05) was found in tissue AA level in different dietary groups. Low amylase, protease, lipase and alkaline phosphatase activities were present in rohu larvae from the mouth opening stage which showed increasing trend with the age of larvae and increasing dietary AA content. A clear dose-dependent modulation of digestive enzyme activities in response to 10%, 20% and 30% AP enriched zooplankton feeding was evidenced from positive correlations between dietary AA content with magnitude of elevation of enzyme activity in different groups. There were 57, 55, 29.2 and 2 fold increases in amylase activity; 7.35, 7.02, 4.43 and 2.73 fold increases in protease activity; 45.636, 41.50, 19.83 and 13.69 fold increases in lipase activity and 6, 5, 3, and 2 fold increases in alkaline phosphatase activity observed in the 15th day post hatch larvae fed 20%, 30%, 10%AP enriched and normal zooplankton respectively, than 4-day post hatch larvae of the respective groups. Enzyme activities were also positively correlated with specific growth rates of wet weight of rohu larvae at the 15th day post hatch. Increased AA might have played an important role in advancing morphological transformation of the digestive tract, protecting gastric mucosa and accelerating growth by the process of tissue formation, which necessitated the requirement of more nutrient thereby, increasing digestive enzyme activity. The regulatory role of AA in the modulation of different digestive enzymes activity and its physiological consequences of nutrient digestibility and utilization during ontogenesis could be extrapolated for better nutrient management of the larvae.

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.

Sulfonamide drugs binding to the colchicine site of tubulin: thermodynamic analysis of the drug-tubulin interactions by isothermal titration calorimetry.

The discovery of several sulfonamide drugs paved the way toward the synthesis of 6 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide, E7010) and 7 (N-(3-fluoro-4-methoxyphenyl)pentafluorobenzenesulfonamide, T138067), both of which inhibit tubulin polymerization and are under clinical development. A series of diarylsulfonamides containing an indole scaffold was also found to have antimitotic properties, but their mode of interactions with tubulin has remained unidentified so far. In this study, we demonstrate that these sulfonamide drugs bind to the colchicine site of tubulin in a reversible manner. They quenched intrinsic tryptophan fluorescence of tubulin presumably due to drug-induced conformational changes in the protein, but were unable to modulate GTPase activity of tubulin in contrast to colchicine that enhances the same enzymatic activity. Further investigation using isothermal titration calorimetry (ITC) revealed that 5 (N-(5-chloro-7-indolyl)-4-methoxybenzenesulfonamide) afforded a large positive value of heat capacity change (DeltaC(p)() = +264 cal mol(-1) K(-1)) on binding to tubulin, suggesting a substantial conformational transition in the protein along with partial enthalpy-entropy compensation. On the other hand, the 2-chloro regioisomer 2 gave a large negative value of DeltaC(p)() (-589 cal mol(-1) K(-1)) along with complete enthalpy-entropy compensation. This thermodynamic profile was thought to be attributable to a prominent contribution of van der Waals interaction and hydrogen bonding between specific groups in the drug-tubulin complex. These results indicate that a mere alteration in the position of a single substituent chlorine on the indole scaffold has a great influence on the drug-tubulin binding thermodynamics.

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.