Browsing by Author "Vikash Kumar Dubey"

Now showing 1 - 20 of 23

Advances in protein misfolding, amyloidosis and its correlation with human diseases
(Springer, 2020) Debanjan Kundu; Kumari Prerna; Rahul Chaurasia; Manoj Kumar Bharty; Vikash Kumar Dubey
Protein aggregation, their mechanisms and trends in the field of neurodegenerative diseases is still far from completely being decoded. It is mainly attributed to the complexity surrounding the interaction between proteins which includes various regulatory mechanisms involved with the presentation of abnormal conditions. Although most proteins are functional in their soluble form, they have also been reported to convert themselves into insoluble aggregates under certain conditions naturally. Misfolded protein forms aggregates which are mostly unwanted by the cellular system and are mostly involved in various pathophysiologies including Alzheimer’s, Type II Diabetes mellitus, Kurus’s etc. Challenges lie in understanding the complex mechanism of protein misfolding and its correlation with clinical evidence. It is often understood that due to the slowness of the process and its association with ageing, timely intervention with drugs or preventive measures will play an essential role in lowering the rate of dementia causing diseases and associated ailments in the future. Today approximately more than 35 proteins have been identified capable of forming amyloids under defined conditions, and nearly all of them have been associated with disease outcomes. This review incorporates a major understanding from the history of diseases associated with protein misfolding, to the current state of neurodegenerative diseases globally, highlighting challenges in drug development and current state of research in a comprehensive manner in the field of protein misfolding diseases. There is increasing clinical association of protein misfolding with regards to amyloids compelling us to thread questions solved and further helping us design possible solutions by generating a pathway-based research on which future work in this field could be driven. © 2020, King Abdulaziz City for Science and Technology.
Biophysical characterization and folding studies of plant protease, wrightin: Identification of folding intermediate under different conditions
(Springer New York, 2009) Ritu Tomar; Vikash Kumar Dubey; M.V. Jagannadham
Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme with major elements of secondary structure being β-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding. Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted. © 2009 Springer Science+Business Media, LLC.
Biophysical characterization of fibroblast growth factor homologous factor-1b (FHF-1b): Sodium dodecyl sulfate promotes two state folding
(Bentham Science Publishers B.V., 2008) Vikash Kumar Dubey; Bishal Kumar Singh; Nandini Sarkar; Monu Pande; Medicherla Venkata Jagannadham
The current article describes the biophysical characterization and folding studies of fibroblast growth factor homologous factor-1b (FHF-1b) in comparison with acidic fibroblast growth factor (FGF-1). Our data indicates that FHF-1 is significantly more stable than FGF-1. The folding mechanism of these two proteins seems to be different although they share high degree of sequence and structural similarity. FHF-1 unfolds through stable intermediate state while unfolding of FGF-1 is two-state. Interestingly, low concentration of sodium dodecyl sulfate (SDS) drives the folding pathway of FHF-1b to two-state. © 2008 Bentham Science Publishers Ltd.
Computational repurposing of potential dimerization inhibitors against sars-cov-2 main protease
(Bentham Science Publishers, 2024) Subhomoi Borkotoky; Archisha Prakash; Gyan Prakash Modi; Vikash Kumar Dubey
Background: The screening, design, and synthesis of various dimerization inhibitors have been an active area of interest for structure-based drug design efforts. Functionally important dimers, such as human immunodeficiency virus (HIV) protease and surviving, are being targeted for such studies over time. Computational repurposing of potential drug candidates provides a cost and time-efficient way in the drug discovery life cycle. Objective: Concerning the current coronavirus disease (COVID-19) scenario, the functionally active dimer of SARS-CoV-2 (severe acute respiratory syndrome) main protease (Mpro) is used as a target to screen possible dimerization inhibitors. Methods: A database of small molecule protein-protein interaction inhibitors was screened for the study. This study used molecular docking, followed by molecular dynamics (MD) simulation and postsimulation binding energy predictions. Results: From the selected 183 compounds, a diazene-based compound and a salicylic-type compound were identified as possible dimerization inhibitors in this study. These two compounds formed stable complexes with the Mpro during the MD simulations. The complexes formed by these two compounds were also unable to form important salt bridge interactions required for the dimerization of the protomers. Conclusion: Experimental studies on both compounds were previously conducted as dimerization inhibitors in HIV. The data led to the possibility of exploring the identified compounds as dimerization inhibitors, which could be important for SARS-CoV-2 therapeutics. © 2024 Bentham Science Publishers.
Differences in the Unfolding of Procerain Induced by pH, Guanidine Hydrochloride, Urea, and Temperature
(2003) Vikash Kumar Dubey; M.V. Jagannadham
The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the α + β class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a β-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.
Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme
(2009) Ritu Tomar; Vikash Kumar Dubey; M.V. Jagannadham
Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (UP) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in UP state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K. © 2009 Elsevier Masson SAS. All rights reserved.
Effect of organic solvents on the molten globule state of procerain: β-Sheet to α-helix switchover in presence of trifluoroethanol
(Bentham Science Publishers B.V., 2006) Vikash Kumar Dubey; Ashu Shah; Medicherla Venkata Jagannadham; Arvind Mohan Kayastha
The effect of methanol and trifluoroethanol (TFE) on the structure and folding of molten globule state of procerain, a cysteine protease from Calotropis procera, was studied by circular dichroism spectroscopy. The magnitude of ellipticity at 215 nm, as a measure of β-sheet content, is dependent on the concentration of the TFE. Interestingly, a switch over from the β-sheet structure of the molten globule state to α-helix was observed at 60% TFE and the ellipticity at 222 nm increased as a function of TFE concentration beyond this critical TFE concentration. Temperature induced unfolding of the molten globule state of procerain in 10% methanol showed stabilization of α-rich domain with concomitant destabilization of β-rich domain. Using higher concentration of methanol (20-40 %) had no stabilizing effect on the α-rich domain however, the β-rich domain was destabilized, indicating that the stability of the domains were not interdependent and that a low concentration of methanol induced stabilization in α-rich domain. © 2006 Bentham Science Publishers Ltd.
Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani
(2013) Sudipta Hazra; Subhalakshmi Ghosh; Madhushree Das Sarma; Smriti Sharma; Mousumi Das; Prakash Saudagar; Vijay Kumar Prajapati; Vikash Kumar Dubey; Shyam Sundar; Banasri Hazra
World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC50~20.7μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC50~7.2μM) as compared to diospyrin (IC50~12.6μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC50~0.18μM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR. © 2013 Elsevier Inc.
IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis
(Academic Press Inc., 2021) Sunita Yadav; Jay Prakash; Om Prakash Singh; Mallikarjuna Rao Gedda; Shashi Bhushan Chauhan; Shyam Sundar; Vikash Kumar Dubey
Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2–3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL. © 2020 Elsevier Inc.
In vivo assessment of antileishmanial property of 4-(4,4,8-trimethyl-7-oxo- 3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester, an oxabicyclo[ 3.3.1]nonanones
(Bentham Science Publishers B.V., 2014) Prakash Saudagar; Shyam Lal Mudavath; Pipas Saha; Anil K. Saikia; Shyam Sundar; Vikash Kumar Dubey
The high toxicity and the growing resistance are the major drawbacks of available antileishmanials. Our previous in vitro studies have identified oxabicyclo[3.3.1]nonanones as antileishmanial agents that act on the redox enzymes of the parasite. In the current study, antileishmanial activity of 4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester (PS 203) the most potent oxabicyclo[3.3.1]nonanone identified in our previous study is evaluated using the hamster model. There was 77.29 ± 3.0 % inhibition of parasite growth observed after a 5-day treatment of 5 mg/kg body weight dose. Further, the in vivo toxicity study of the compound in Swiss albino mice revealed no hepatic or renal toxicity. © 2014 Bentham Science Publishers.
Insights into pH-induced conformational transition of β-galactosidase from pisum sativum leading to its multimerization
(2010) Alka Dwevedi; Vikash Kumar Dubey; Medicherla V. Jagannadham; Arvind M. Kayastha
Although β-galactosidases are physiologically a very important enzyme and have may therapeutics applications, very little is known about the stability and the folding aspects of the enzyme. We have used β-galactosidase from Pisum sativum (PsBGAL) as model system to investigate stability, folding, and function relationship of β-galactosidases. PsBGAL is a vacuolar protein which has a tendency to multimerize at acidic pH with protein concentration ≥100 μg mL-1 and dissociates into its subunits above neutral pH. It exhibits maximum activity as well as stability under acidic conditions. Further, it has different conformational orientations and core secondary structures at different pH. Substantial predominance of β-content and interfacial interactions through Trp residues play crucial role in pH-dependent multimerization of enzyme. Equilibrium unfolding of PsBGAL at acidic pH follows four-state model when monitored by changes in the secondary structure with two intermediates: one resembling to molten globule-like state while unfolding seen from activity and tertiary structure of PsBGAL fits to two-state model. Unfolding of PsBGAL at higher pH always follows two-state model. Furthermore, unfolding of PsBGAL reveals that it has at least two domains: α/β barrel containing catalytic site and the other is rich in β-content responsible for enzyme multimerization. © 2010 Springer Science+Business Media, LLC.
Leishmania donovani evades Caspase 1 dependent host defense mechanism during infection
(Elsevier B.V., 2019) Gundappa Saha; Bakulesh M. Khamar; Om Prakash Singh; Shyam Sundar; Vikash Kumar Dubey
Significant advances have been made in understanding the regulation of inflammasomes and its involvement in innate immunity during pathogenic infections. Inflammasome activation is a tightly regulated process that provides defense against pathogenic infection and important for inflammatory response. Very few studies on the involvement of NLRP3 inflammasome protein complex have been reported in leishmanial infections with contradictory results and without much mechanistic insights. However, the role of NLRP3 inflammasome and its components has not been well deciphered in Leishmania donovani infection. Here we report for the first time a detailed mechanism and plausible impairment of caspase 1 activation during L. donovani infection leading to the survival of these parasites inside the host cells. Low mRNA expression of pro-caspase 1 and lack of caspase 1 maturation were observed after infection, hindering the processing of pro-IL-1β and pro-IL-18 into their mature counter parts. Further, siRNA mediated knock-down of caspase 1 in macrophage cells (THP-1) resulted in significantly higher parasitic burden validating the importance of caspase 1 in the host defense mechanism. Taken together, our data suggests that the parasite inhibits caspase 1 activation to evade the inflammatory nature of pyroptosis. © 2018
Ligand conjugated lipid-based nanocarriers for cancer theranostics
(John Wiley and Sons Inc, 2022) Rahul Kumar; Daphika S. Dkhar; Rohini Kumari; Divya; Supratim Mahapatra; Ananya Srivastava; Vikash Kumar Dubey; Pranjal Chandra
Cancer is one of the major health-related issues affecting the population worldwide and subsequently accounts for the second-largest death. Genetic and epigenetic modifications in oncogenes or tumor suppressor genes affect the regulatory systems that lead to the initiation and progression of cancer. Conventional methods, including chemotherapy/radiotherapy/appropriate combinational therapy and surgery, are being widely used for theranostics of cancer patients. Surgery is useful in treating localized tumors, but it is ineffective in treating metastatic tumors, which spread to other organs and result in a high recurrence rate and death. Also, the therapeutic application of free drugs is related to substantial issues such as poor absorption, solubility, bioavailability, high degradation rate, short shelf-life, and low therapeutic index. Therefore, these issues can be sorted out using nano lipid-based carriers (NLBCs) as promising drug delivery carriers. Still, at most, they fail to achieve site-targeted drug delivery and detection. This can be achieved by selecting a specific ligand/antibody for its cognate receptor molecule expressed on the surface of the cancer cells. In this review, we have mainly discussed the various types of ligands used to decorate NLBCs. A list of the ligands used to design nanocarriers to target malignant cells has been extensively undertaken. The approved ligand-decorated lipid-based nanomedicines with their clinical status have been explained in tabulated form to provide a wider scope to the readers regarding ligand-coupled NLBCs. © 2022 Wiley Periodicals LLC.
Molecular docking and in vitro antileishmanial evaluation of chromene-2-thione analogues
(2012) Rajiv Kumar Verma; Vijay Kumar Prajapati; Girijesh Kumar Verma; Deblina Chakraborty; Shyam Sundar; Madhukar Rai; Vikash Kumar Dubey; Maya Shankar Singh
Leishmaniases are an epidemic in various countries, and the parasite is developing resistance against available drugs. Thus, development of new drugs against Leishmania is an open area of investigation for synthetic organic chemists. To meet this challenge, a series of chromene-2-thione derivatives have been synthesized and docked into the active site of trypanothione reductase (TryR) enzyme required for redox balance of the parasite. These were screened on promastigote, axenic amastigote, and intracellular amastigote stages of Leishmania donovani and found to show high levels of antileishmanial activity together with minimal toxicity to human peripheral blood mononuclear cells. Compounds 3b and 3k were found to be the most active among the tested compounds. Although the compounds show moderate antileishmanial activity, they identify a chemical space to design and develop drugs based on these chromene-2-thione derivatives against the Leishmania parasite. © 2012 American Chemical Society.
Procerain, a stable cysteine protease from the latex of Calotropis procera
(Elsevier Ltd, 2003) Vikash Kumar Dubey; M.V. Jagannadham
A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 °C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not L-Ala-Ala-p-nitroanilide, L-Ala p-nitroanilide and N-d-Benzoyl-DL-Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (ε280nm1%) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 °C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue. © 2003 Elsevier Science Ltd. All rights reserved.
Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain
(2010) Abhay Narain Singh; Anil Kumar Shukla; M.V. Jagannadham; Vikash Kumar Dubey
Proteases have applications in food, detergent and pharmaceutical industries. A novel protease has been purified from the latex of Calotropis procera and characterized. As another cysteine protease, procerain, is reported from the same source, the newly purified enzyme was named as procerain B. The enzyme shows distinct properties compared to procerain, in terms of cleavage recognition site, immunological properties and other physical properties like molecular weight, isoelectric point, etc. The newly purified enzyme shows a broad optimum pH (6.5-8.5) as well as broad optimum temperature (40-60 °C). Additionally, the enzyme retains its activity where most of other proteases are not active. Moreover, the enzyme appeared to be very efficient in hydrolysis of blood stain and may have potential application in detergent industries. Simple and economic purification of procerain B, together with easy availability of latex, makes the large-scale production of procerain B possible, thus enables to explore various industrial as well as biotechnological applications. © 2009 Elsevier Ltd. All rights reserved.
Repurposing the in-house generated Alzheimer’s disease targeting molecules through computational and preliminary in-vitro studies for the management of SARS-coronavirus-2
(Springer Nature, 2024) Gourav Singh; Jobin Thomas; Sahil Wadhawa; Anurag Kashyap; Syed Ajijur Rahaman; Subhomoi Borkotoky; Agnisha Datta; Gireesh Kumar Singh; Indubhusan Mishra; Geeta Rai; Jitendra Satija; Vikash Kumar Dubey; Gyan Modi
Covid-19 was declared a world pandemic. Recent studies demonstrated that Covid-19 impairs CNS activity by crossing the blood–brain barrier and ensuing cognitive impairment. In this study, we have utilized Covid-19 main protease (Mpro) as a biological target to repurpose our previously reported multifunctional compounds targeting Alzheimer’s disease. Molecular docking, spatial orientation, molecular dynamics simulation, MM-GBSA energy calculation, and DFT studies were carried out with these molecules. Among all the compounds, F27, F44, and F56 exhibited higher binding energy (− 8.03, − 8.65, and − 8.68 kcal/mol, respectively) over the co-crystal ligand O6K (− 7.00 kcal/mol). In MD simulation, compounds F27, F44, and F56 could make a stable complex with Mpro target throughout the simulation. The compounds were synthesized following reported methods and subjected for cytotoxicity, and assessment of their capability to cross the blood–brain barrier in PAMPA assay, and antioxidant property evaluation through DPPH assay. The compounds F27, F44, and F56 exhibited cytocompatibility with the SiHA cell line and also displayed significant antioxidant properties with IC50 = 45.80 ± 0.27 μM, 44.42 ± 0.30 μM, and 42.74 ± 0.23 μM respectively. In the PAMPA assays, the permeability coefficient (Pe) value of F27, F44, and F56 lies in the acceptable range (Pe > 4). The results of the computational and preliminary in-vitro studies strongly corroborate the potential of F27, F44, and F56 as a lead for further optimization in treating the CNS complications associated with Covid-19. © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023.
SDS induced refolding of pre-molten globule state of cryptolepain: Differences in chemical and temperature-induced equilibrium unfolding of the protein in SDS-induced state
(2013) N.K. Prasanna Kumari; Vikash Kumar Dubey; Medicherla V. Jagannadham
Interaction of sodium dodecyl sulfate (SDS) with a globular protein cryptolepain to induce stabilization of the pre-molten globule state of the protein under acidic condition was studied. At pH 2.0, cryptolepain is in partially unfolded state with native like secondary structure, exposed hydrophobic surfaces without any rigid tertiary structure. Unfolding of cryptolepain in the pre-molten globule state (state at pH 2.0) is co-operative suggesting that the protein unfolds as a single entity. Addition of SDS below its critical micelle concentration of 2.0 mM at acidic pH drives the protein to a non-native state similar to molten globule state seen with other proteins. Temperature induced unfolding of such SDS-induced state of the cryptolepain is biphasic suggesting that the molecular structure of the protein is constituted of two structural entities which are stabilized differentially in the presence of SDS and unfold sequentially. Whereas the chemical induced unfolding of SDS-induced state of cryptolepain is co-operative with higher transition midpoints indicating that the protein is stabilized as a whole. Unfolding of SDS-induced state of cryptolepain is very similar to the unfolding of the cryptolepain at very low pH (0.5), where the protein exists in molten globule state. Thus, SDS interactions result in the refolding of the acid unfolded state of the protein and drive the protein into a non-native state similar to the molten-globule state seen at very low pH. © 2012 The National Academy of Sciences, India.
Snapshots of protein folding problem: Implications of folding and misfolding studies
(2006) Vikash Kumar Dubey; Monu Pande; M.V. Jagannadham
Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed. © 2006 Bentham Science Publishers Ltd.
Stability and unfolding studies on alkaline denatured state (Ip) of pepsin
(2009) Monu Pande; N.K. Prasanna Kumari; Vikash Kumar Dubey; Pinky Tripathi; M.V. Jagannadham
Pepsin exists as alkaline denatured state (Ip) in pH range 8-10, where the N-terminal domain of the protein is mostly unfolded while the C-terminal domain is intact. The effects of fluorinated (TFE) and non-fluorinated (methanol) organic solvents on this partially unfolded state (Ip) of pepsin were investigated using various spectroscopic methods. Both, fluorinated (TFE) and non-fluorinated (methanol) organic solvents induce secondary structure (α-helix) after a critical concentration. The Ip state of pepsin unfolds in cooperative manner but the transition was found to be non-cooperative in the presence of 40% methanol or TFE. The differences in the unfolding of the protein in the presence and the absence of these organic solvents were interpreted. Our results indicate that unfolding transitions in Ip state are mostly dominated by unfolding of C-terminal domain because the N-terminal domain is largely unstructured in this state. The organic solvents (TFE and methanol) induce more secondary structure in N-terminal domain and make it another unfolding entity with different stability compare to C-terminal resulting into sequential unfolding of the domain. © 2009 Elsevier Ltd. All rights reserved.