Publications by authors named "Purnananda Guptasarma"

54 Publications

N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate.

Biochemistry 2021 Jun 21;60(23):1836-1852. Epub 2021 May 21.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India.

HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in within which the early, late, and stationary phases of growth are dominated by HU-AA, HU-BB, and HU-AB dimers, respectively. Here, using genetic manipulation, mass spectrometry, spectroscopy, chromatography, and electrophoretic examination of glutaraldehyde-mediated cross-linking of subunits, in combination with experiments involving mixing, co-expression, unfolding, and refolding of HU chains, we show that the spontaneous formation of HU-AB heterodimers that is reported to occur upon mixing of wild-type HU-AA and HU-BB homodimers does not occur if chains possess N-terminal extensions. We show that N-terminal extensions interfere with the conversion of homodimers into heterodimers. We also show that heterodimers are readily formed at anticipated levels by chains possessing N-terminal extensions , when direct chain-chain interactions are facilitated through production of HU-A and HU-B chains from proximal genes located upon the same plasmid. From the data, two explanations emerge regarding the mechanism by which N-terminal extensions happen to adversely affect the conversion of homodimers into heterodimers. (1) The disappearance of the α-amino group at HU's N-terminus impacts the intersubunit stacking of β-sheets at HU's dimeric interface, reducing the ease with which subunits dissociate from each other. Simultaneously, (2) the presence of an N-terminal extension appears to sterically prevent the association of HU-AA and HU-BB homodimers into a critically required, heterotetrameric intermediate (within which homodimers could otherwise exchange subunits without releasing monomers into solution, by remaining physically associated with each other).
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http://dx.doi.org/10.1021/acs.biochem.1c00081DOI Listing
June 2021

HU-AB simulacrum: Fusion of HU-B and HU-A into HU-B-A, a functional analog of the Escherichia coli HU-AB heterodimer.

Biochem Biophys Res Commun 2021 Jun 5;560:27-31. Epub 2021 May 5.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

In enteric bacteria such as Escherichia coli, there are two homologs of the DNA-binding nucleoid associated protein (NAP) known as HU. The two homologs are known as HU-A and HU-B, and exist either in the form of homodimers (HU-AA, or HU-BB) or as heterodimers (HU-AB), with different propensities to form higher-order oligomers. The three different dimeric forms dominate different stages of bacterial growth, with the HU-AB heterodimer dominating cultures in the stationary phase. Due to similarities in their properties, and the facile equilibrium that exists between the dimeric forms, the dimers are difficult to purify away from each other. Although HU-AA and HU-BB can be purified through extensive ion-exchange chromatography, reestablishment of equilibrium interferes with the purification of the HU-AB heterodimer (which constitutes ∼90% of any population with equal numbers of HU-B and HU-A chains). Here, we report the creation of a functional analog of HU-AB that does not appear to partition to generate any minority populations of HU-AA or HU-BB. The analog was constructed through genetic fusion of the HU-B and HU-A chains into a single polypeptide (HU-B-A) with a glycine/serine-rich linker of 11 amino acids separating HU-B from HU-A, and a histidine tag at the N-terminus of HU-B. HU-B-A folds to bind 4-way junction DNA, and displays a significant tendency to form dimers (i.e., analogs of HU tetramers), and a higher thermodynamic stability than HU-BB or HU-AA, thus explaining why it dominates mixtures of HU-B and HU-A chains.
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http://dx.doi.org/10.1016/j.bbrc.2021.04.107DOI Listing
June 2021

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms.

J Biol Chem 2021 Mar 10:100532. Epub 2021 Mar 10.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306. Electronic address:

In biofilms, bacteria that possess a negatively-charged surface are embedded within a matrix of polymers consisting mainly of negatively-charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively-charged substance, e.g., a basic protein, exists within biofilms to neutralize charge-charge repulsions and act as a 'glue' attaching negatively-charged bacteria to negatively-charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly-abundant nucleoid-associated protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, e.g., (a) availability in association with e-DNA; (b) multivalent DNA-binding; (c) non-sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU-cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and non-canonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical-chemical, microscopy-based and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS; that this facilitates DNA-DNA, DNA-LPS and LPS-LPS interactions; and that this facilitates bacterial clumping as well as attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (non-shaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide and move away from each other, in opposition to the accreting influence of HU).
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http://dx.doi.org/10.1016/j.jbc.2021.100532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063757PMC
March 2021

A novel protein-engineered dsDNA-binding protein (HU-Simulacrum) inspired by HU, a nucleoid-associated DNABII protein.

Biochem Biophys Res Commun 2021 01 10;534:47-52. Epub 2020 Dec 10.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.088DOI Listing
January 2021

An ultra-stable glucanotransferase-cum-exoamylase from the hyperthermophile archaeon Thermococcus onnurineus.

Arch Biochem Biophys 2019 04 4;665:114-121. Epub 2019 Mar 4.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Knowledge City, Sector-81, SAS Nagar, Mohali, Punjab, 140306, India. Electronic address:

The genome of the hyperthermophile archaeon Thermococcus onnurineus (strain NA1) encodes a 652 residues-long putative 4-α-glucanotransferase of the GH 57 family which we have expressed in Escherichia coli. The enzyme (TonAmyGT) appears to remove glucose from the reducing end of a donor glucan and transfers it to the non-reducing end of an acceptor glucan, creating a pool of oligosaccharides through disproportionation of any substrate maltooligosaccharide, with maltose acting substantively as the smallest donor glucan as well as the smallest acceptor glucan. Additionally, glucose is also cleaved from maltooligosaccharides and released into solution without being transferred to an acceptor, causing the enzyme to function as an exo-amylase (which can digest starch) in addition to its activity as a glucanotransferase. TonAmyGT functions over a broad range of temperature (20-100 °C) and pH (4.0-9.0), and shows extreme resistance to chemical and thermal denaturation, displaying a melting temperature of 104 °C, at a pressure of 35 psi, in a differential scanning calorimeter. An interesting characteristic is that the glucanotransferase activity shows feedback inhibition through glucose (which the enzyme itself generates), indicating that the exo-amylase and glucanotransferase activities regulate each other.
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http://dx.doi.org/10.1016/j.abb.2019.02.017DOI Listing
April 2019

Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins.

Electrophoresis 2019 05 12;40(9):1273-1281. Epub 2019 Feb 12.

Centre for Protein Science, Design and Engineering, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Punjab, India.

During SDS-PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues-long, sequence-homologous, structurally-analogous, extracellular domains of human E- and N-cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively-charged and positively-charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater-than-expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS-protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.
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http://dx.doi.org/10.1002/elps.201800219DOI Listing
May 2019

Structure-guided mutational evidence and postulates explaining how a glycohydrolase from Pyrococcus furiosus functions simultaneously as an amylase and as a 4-α-glucanotransferase.

Biochem Biophys Res Commun 2019 02 11;509(4):892-897. Epub 2019 Jan 11.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

Pyrococcus furiosus exoamylase-cum-4-α-glucanotransferase (4-α-GTase; PF0272; PfuAmyGT) is reported to both (i) act upon starch, and (ii) catalyze 'disproportionation' of maltooligosaccharides (with glucose as the smallest product). PfuAmyGT shares ∼65% sequence identity with a homo-dimeric Thermococcus litoralis 4-α-GTase, for which structures are available in complex with a non-hydrolysable analog of maltotetraose (acarbose) bound to one subunit and maltose (of unknown origin) bound to the other subunit. We structurally transposed the maltose onto the acarbose-bound subunit and discovered that the two molecules lie juxtaposed in what could be perfect 'acceptor' and 'donor' substrate-binding sites, respectively. We also discovered that there is a loop between the two sites which could use an available aspartate to excise a glucose from the donor, and an available tryptophan to transfer the glucose to the non-reducing end of the acceptor glucan. We derived a structure for PfuAmyGT through homology-based modeling, identified the potential donor site, acceptor site, glucan-transferring loop, and catalytically important residues, and mutated these to alanine to examine effect(s) upon activity. Mutation D362A abolished creation of shorter, or longer, maltooligosaccharides. Mutation W365A abolished creation of longer oligosaccharides. Mutation H366A had no effect on activity. We propose that D362 facilitates glucose excision, and that W365 facilitates its transfer, either (a) directly into solution (allowing PfuAmyGT to act as an exoamylase), or (b) by glycoside bond formation with an acceptor (allowing PfuAmyGT to act as a 4-α-glucanotransferase), depending upon whether the acceptor site is vacant or occupied in a reaction cycle.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.021DOI Listing
February 2019

Structural-Mechanical and Biochemical Functions of Classical Cadherins at Cellular Junctions: A Review and Some Hypotheses.

Adv Exp Med Biol 2018;1112:107-138

Centre for Protein Science, Design and Engineering, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India.

This article begins with a general review of cell adhesion molecules (CAMs) and narrows the focus down progressively to the cadherins (calcium binding-dependent CAMs), classifications of subfamilies of the cadherins, type I (E- and N-) cadherins, evolutionary relationships amongst cadherins, structural-mechanical and functional consequences of calcium binding to the cadherins, differential molecular interactions involving the extracellular (ecto) and intracellular (cytoplasmic) domains of the cadherins, multiple adherence-related homophilic and heterophilic interactions and associated functions of E- and N-cadherin in organismal development and disease and cadherin trafficking and membrane rafts. It ends by summarizing multiple perspectives and hypotheses concerning different aspects of cadherin structure, stability and function.
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http://dx.doi.org/10.1007/978-981-13-3065-0_9DOI Listing
July 2019

Multiple thermostable enzyme hydrolases on magnetic nanoparticles: An immobilized enzyme-mediated approach to saccharification through simultaneous xylanase, cellulase and amylolytic glucanotransferase action.

Int J Biol Macromol 2018 Dec 22;120(Pt B):1650-1658. Epub 2018 Sep 22.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector-81, SAS, Nagar, Punjab 140306, India. Electronic address:

Microbe-derived enzymes such as xylanases, cellulases and amylases, are efficient at hydrolyzing plant biomass. Efforts to harness the functionalities of these enzymes towards applications in energy and fuel biosciences, and food and nutrition, continue apace in many laboratories. Given that enzymes derived from mesophile proteomes undergo facile denaturation and/or degradation at ambient temperatures, and require frequent replenishment during bioprocessing, it is desirable that they be replaced by structurally-stable enzymes capable of functioning efficiently and resisting denaturation and degradation, immobilized on solid media to further add to stability and facilitate recovery and reuse. Towards these objectives, we used synthetic magnetic nanoparticles (MNPs) and immobilized upon their surfaces three different structurally-stable hydrolases: a thermostable xylanase (BSX) derived from Bacillus sp. NG-27, a cellulase (RMCel12A) derived from Rhodothermus marinus, and an amylase-cum-glucanotransferase (PfuAmyGT) derived from Pyrococcus furiosus. The MNPs were activated with glutaraldehyde and BSX, RMCel12A, and PfuAmyGT, respectively, were covalently immobilized with efficiencies of ~92%, 45% and 93%. The enzymes and the MNPs were fully characterized before and after immobilization, and the immobilized enzymes were found to be active at 50 °C against synthetic substrates as well as pre-treated biomass derived from corn cob and rice husk. The enzyme-coupled MNPs displayed high stability upon storage properties, high operational stability as well as high reusability (retaining 69, 48, and 50% residual activity after 13 uses for BSX, RMCel12A and PfuAmyGT, respectively). Experiments were also conducted with MNPs loaded simultaneously with all three enzymes. Such immobilized enzyme combinations on MNPs can be used in the saccharification of plant biomass.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.09.106DOI Listing
December 2018

Probing the excited state dynamics of Venus: origin of dual-emission in fluorescent proteins.

Faraday Discuss 2018 04;207(0):39-54

Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector 81, SAS Nagar, Mohali, Punjab 140306, India.

Fluorescent proteins exhibit interesting excited state photochemistry, leading to bright fluorescence emission that renders their versatile biological role and wide use as biomarkers. A molecular-level mechanism of the excited state dynamics is desirable to pinpoint the origin of the bright fluorescence of these proteins. Here we present studies on a yellow fluorescent protein variant, Venus, and investigate the photophysics behind the dual fluorescence emission upon UV excitation. Based on our studies, we propose that the unique nature of the potential energy surface is responsible for the observation of minor fluorescence in Venus which is not seen in wild type GFP.
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http://dx.doi.org/10.1039/c7fd00187hDOI Listing
April 2018

Characterization of a mildly alkalophilic and thermostable recombinant Thermus thermophilus laccase with applications in decolourization of dyes.

Biotechnol Lett 2018 Feb 23;40(2):285-295. Epub 2017 Oct 23.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Knowledge City, Sector-81, SAS Nagar, Mohali, Punjab, 140306, India.

Objective: To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli.

Result: Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K ) and maximum velocity (V) at 60 °C and pH 6.0 were determined to be 2.4 × 10 µM and 0.04 × 10 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme's ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS.

Conclusions: Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.
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http://dx.doi.org/10.1007/s10529-017-2461-8DOI Listing
February 2018

Endoglucanase activity at a second site in triosephosphate isomerase-Promiscuity or compensation for a metabolic handicap?

FEBS Open Bio 2017 08 11;7(8):1126-1143. Epub 2017 Jul 11.

Department of Biological Sciences Centre for Protein Science Design and Engineering (CPSDE) Indian Institute of Science Education and Research (IISER) Mohali Punjab.

The eight-stranded (β/α) barrel fold known as the Triosephosphate isomerase (TIM) barrel is the most commonly observed fold in enzymes, displaying an eightfold structural symmetry. The sequences and structures of different TIM barrel enzymes suggest that nature exploits the modularity inherent in the eightfold symmetry to generate enzymes with diverse enzymatic activities and, in certain cases, more than one catalytic activity per enzyme. Here, we report the discovery, verification, and characterization of such an additional activity, a novel endoglucanase/cellulase activity in what is otherwise a triosephosphate isomerase from the hyperthermophile archaeon (PfuTIM). The activity is seen in two different ranges of temperatures, with one maximum at 40 °C and a second maximum close to 100 °C. The endoglucanase/cellulase activity is inhibited by norharman, a TIM inhibitor, which is suspected to bind at a site different to that of the regular substrate, glyceraldehyde-3-phosphate (G3P). However, endoglucanase/cellulose activity is not inhibited either by G3P analogs or by glycine-scanning mutations involving residues in loops 1, 4, and 6 of PfuTIM, which are known to be important for TIM activity. It appears, therefore, that two different sites on PfuTIM are responsible for the observed TIM and endoglucanase activities. We discuss possible correlations between this discovery and certain unusual features of the glycolytic pathway in .

Enzyme: Triosephosphate isomerase (EC:5.3.1.1).
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http://dx.doi.org/10.1002/2211-5463.12249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537068PMC
August 2017

Creation of active TIM barrel enzymes through genetic fusion of half-barrel domain constructs derived from two distantly related glycosyl hydrolases.

FEBS J 2016 12 10;283(23):4340-4356. Epub 2016 Nov 10.

Department of Biological Sciences, Centre for Protein Science, Design and Engineering, Indian Institute of Science Education and Research (IISER), Mohali, SAS Nagar, India.

Diverse unrelated enzymes that adopt the beta/alpha (or TIM) barrel topology display similar arrangements of beta/alpha units placed in a radial eight-fold symmetry around the barrel's axis. The TIM barrel was originally thought to be a single structural domain; however, it is now thought that TIM barrels arose from duplication and fusion of smaller half-barrels consisting of four beta/alpha units. We describe here the design, expression and purification, as well as characterization of folding, activity and stability, of chimeras of two TIM barrel glycosyl hydrolases, made by fusing different half-barrel domains derived from an endoglucanase from Clostridium cellulolyticum, CelCCA and a beta-glucosidase from Pyrococcus furiosus, CelB. We show that after refolding following purification from inclusion bodies, the two half-barrel fusion chimeras (CelCCACelB and CelBCelCCA) display catalytic activity although they assemble into large soluble oligomeric aggregated species containing chains of mixed beta and alpha structure. CelBCelCCA displays hyperthermophile-like structural stability as well as significant stability to chemical denaturation (C of 2.6 m guanidinium hydrochloride), whereas CelCCACelB displays mesophile-like stability (T of ~ 71 °C). The endoglucanase activities of both chimeras are an order of magnitude lower than those of CelB or CelCCA, whereas the beta-glucosidase activity of CelBCelCCA is about two orders of magnitude lower than that of CelB. The chimera CelCCACelB shows no beta-glucosidase activity. Our results demonstrate that half-barrel domains from unrelated sources can fold, assemble and function, with scope for improvement.

Enzyme: Pyrococcus furiosus beta-glucosidase (CelB, EC: 3.2.1.21). Clostridium cellulolyticum endoglucanase A (CelCCA, EC: 3.2.1.4).
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http://dx.doi.org/10.1111/febs.13927DOI Listing
December 2016

Arsenic and 17-β-estradiol bind to each other and neutralize each other's signaling effects.

Biochem Biophys Res Commun 2016 09 23;477(4):575-580. Epub 2016 Jun 23.

Center for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India. Electronic address:

We report that arsenic trioxide (ATO) and 17-beta-estradiol (E2) abolish each other's independent cell signaling effects in respect of cell survival and proliferation/migration of breast cancer (MCF-7) cells. The possibility that this is due to binding of ATO to E2 was confirmed through difference absorption spectroscopy, chromatography-coupled voltammometry and 1-D (1)H and (13)C NMR spectroscopy. Binding leads to attenuation of E2's hydroxyl (1)H peaks at its C17 and C3 carbon positions. The results suggest that ATO and E2 can titrate each other's levels, potentially explaining why sustained arsenic exposure tends to be associated with delays in age of menarche, advanced age of menopause, poorer sperm quality, higher overall morbidity in men, and lower incidences of breast cancer in women in some arsenic-contaminated areas.
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http://dx.doi.org/10.1016/j.bbrc.2016.06.087DOI Listing
September 2016

The Achilles' Heel of "Ultrastable" Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge.

Biochemistry 2016 07 6;55(28):3920-36. Epub 2016 Jul 6.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali , Knowledge City, Sector 81, SAS Nagar, Punjab, India 140306.

Low concentrations (<3.0 mM) of the anionic surfactant sodium dodecyl sulfate (SDS) have been shown to induce the formation of amyloid fibers in more than 20 different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be "ultrastable" and inordinately resistant to structural perturbations at room temperature. Here, we show that low (<4.5 mM) concentrations of SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative circular dichroism band at ∼230 nm. These effects are not seen if the proteins have a net negative charge or when higher concentrations of SDS are used (which induce helix formation instead). Our results appear to reveal a potential weakness or "Achilles' heel" in ultrastable proteins from hyperthermophiles. They also provide very strong support for the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability.
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http://dx.doi.org/10.1021/acs.biochem.5b01343DOI Listing
July 2016

Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.

Proteins 2015 10;83(10):1813-22

CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.

Although unglycosylated HuEpo is fully functional, it has very short serum half-life. However, the mechanism of in vivo clearance of human Epo (HuEpo) remains largely unknown. In this study, the relative importance of protease-sensitive sites of recombinant HuEpo (rHuEpo) has been investigated by analysis of structural data coupled with in vivo half-life measurements. Our results identify α3-α4 inter-helical loop region as a target site of lysosomal protease Cathepsin L. Consistent with previously-reported lysosomal degradation of HuEpo, these results for the first time identify cleavage sites of rHuEpo by specific lysosomal proteases. Furthermore, in agreement with the lowered exposure of the peptide backbone around the cleavage site, remarkably substitutions of residues with bulkier amino acids result in significantly improved in vivo stability. Together, these results have implications for the mechanism of in vivo clearance of the protein in humans.
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http://dx.doi.org/10.1002/prot.24865DOI Listing
October 2015

Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry.

Anal Biochem 2015 Nov 21;488:6-8. Epub 2015 Jul 21.

Center for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

Mass spectrometry-based amino acid sequencing is currently based almost entirely on collision-induced peptide fragmentation and analyses. Here, we describe a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, heat-loving Bacillus subtilis-derived aminopeptidase (BsuAP), which acts optimally at 70 °C and allows 'single-shot' sequencing to be carried out through simultaneous accumulation, and detection of sub-populations of peptides of progressively reducing length.
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http://dx.doi.org/10.1016/j.ab.2015.07.006DOI Listing
November 2015

Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system.

Anal Biochem 2015 Sep 14;484:180-2. Epub 2015 Jun 14.

Center for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India. Electronic address:

Extremely low levels of "leaky" expression of genes in bacterial protein expression systems can severely curtail cell viability when expressed proteins are toxic. A general method for sensitive detection of such expression is lacking. Here, we present a method based on microscopic visualization of a fluorescent "reporter" protein (RFP-HU-A) constructed by fusing red fluorescent protein (RFP) to the N-terminus of a nucleoid-associated, histone-like DNA-binding protein, HU-A. Localization of RFP-HU-A within nucleoids facilitates detection, quantitation, and characterization of leaky expression at the single-cell level.
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http://dx.doi.org/10.1016/j.ab.2015.06.011DOI Listing
September 2015

'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

Biochem Biophys Res Commun 2015 May 27;460(3):753-8. Epub 2015 Mar 27.

Centre for Protein Science, Design and Engineering, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector-81, SAS Nagar, Manauli P.O., Punjab, 140306, India. Electronic address:

Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes.
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http://dx.doi.org/10.1016/j.bbrc.2015.03.102DOI Listing
May 2015

Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

PLoS One 2014 22;9(4):e95725. Epub 2014 Apr 22.

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, SAS Nagar, Punjab, India.

Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m) under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum) concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD) varieties. We establish (a) that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b) that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c) that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d) that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT), resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095725PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995793PMC
January 2015

The key to the extraordinary thermal stability of P. furiosus holo-rubredoxin: iron binding-guided packing of a core aromatic cluster responsible for high kinetic stability of the native structure.

PLoS One 2014 6;9(3):e89703. Epub 2014 Mar 6.

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, SAS Nagar, Punjab, India; Protein Engineering Division, Institute of Microbial Technology (IMTECH), Chandigarh, India.

Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089703PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945938PMC
February 2015

Hyperthermophile protein behavior: partially-structured conformations of Pyrococcus furiosus rubredoxin monomers generated through forced cold-denaturation and refolding.

PLoS One 2014 6;9(3):e80014. Epub 2014 Mar 6.

Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar (Mohali), Punjab, India; Protein Science & Engineering Division, Institute of Microbial Technology, (IMTECH), Council of Scientific & Industrial Research (CSIR), Chandigarh, India.

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as 'Trishanku' unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native 'Trishanku' refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945965PMC
February 2015

Introduction of a thermophile-sourced ion pair network in the fourth beta/alpha unit of a psychophile-derived triosephosphate isomerase from Methanococcoides burtonii significantly increases its kinetic thermal stability.

Biochim Biophys Acta 2013 Jun 14;1834(6):1023-33. Epub 2013 Jan 14.

Department of Protein Science and Engineering, Institute of Microbial Technology, Chandigarh, India.

Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (β/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (β/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70°C, when a temperature scanning rate of 90°C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~10°C when the temperature scanning rate is lowered to 5°C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4-6°C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (β/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions.
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http://dx.doi.org/10.1016/j.bbapap.2013.01.001DOI Listing
June 2013

Insufficient (sub-native) helix content in soluble/solid aggregates of recombinant and engineered forms of IL-2 throws light on how aggregated IL-2 is biologically active.

Protein J 2012 Oct;31(7):529-43

Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.

Interleukin 2 (IL-2) is an extremely aggregation-prone, all-alpha helical cytokine. In its receptor-bound state, ~72 % of the polypeptide chain adopts helical structure and there is no beta sheet content whatsoever. In the past, recombinant IL-2 has been formulated and used therapeutically in humans, following production in E. coli. Therapeutic IL-2 consists entirely of functionally-active soluble aggregates with ~30 subunits per aggregate particle. Side-effects attributed to aggregation resulted in discontinuation of usage over a decade ago. Structurally, and biochemically, activity in IL-2 aggregates can potentially be explained in one of two ways : (a) individual IL-2 chains exist in sterically-accessible, receptor binding-competent (native) structures, allowing aggregates to bind directly to IL-2 receptors (IL-2R); alternatively, (b) IL-2 chains dissociate from aggregates, become free to adopt native structure, and then bind to IL-2R. We produced native IL-2 and numerous engineered forms in E. coli with the objective of obtaining insights into these possibilities. Each IL-2 variant was subjected to size exclusion chromatography, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). All forms produced and studied (including those with native IL-2 sequences) turned out to aggregate and also display less than ~50 % helix content as well as significant beta sheet content. No conditions were found that obviate aggregation. Aggregated IL-2 is thus insufficiently native-like to bind to IL-2R. Activity in aggregates thus probably owes to adoption of receptor binding-competent structures by chains that have already dissociated from aggregates.
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http://dx.doi.org/10.1007/s10930-012-9429-2DOI Listing
October 2012

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion.

Anal Biochem 2011 Jan 17;408(2):263-8. Epub 2010 Sep 17.

Protein Science & Engineering, Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India.

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.
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http://dx.doi.org/10.1016/j.ab.2010.09.013DOI Listing
January 2011

Structures of differently aggregated and precipitated forms of gamma B crystallin: an FTIR spectroscopic and EM study.

Protein Pept Lett 2010 Sep;17(9):1155-62

Department of Protein Science & Engineering, Institute of Microbial Technology (IMTECH), Chandigarh 160036, India.

The lens protein, gamma B-crystallin, precipitates during cataract formation. As a recombinant protein, in aqueous solution, gamma B aggregates and precipitates upon heating, cooling, exposure to ultraviolet light, or refolding from a denatured state. We have studied soluble gamma B crystallin, as well as each of the above aggregated forms, to determine whether gamma B's polypeptide chain is differently organized in each form. For this purpose, we used : (a) Fourier Transform Infra Red (FTIR) spectroscopy in the horizontal attenuated total reflectance (HATR) mode, to examine changes in secondary structural content, and (b) transmission electron microscopy (TEM) to examine gross morphological differences. The peak of the gamma B FTIR amide I band shifts from approximately 1633 cm(-1) to approximately 1618 cm(-1) in heat-, UV- and refolding-induced gamma B precipitates, indicating that narrow beta sheets with fewer strands and higher strand twist angles are becoming reorganized into wider, more planar sheets containing larger numbers of shorter strands, with smaller twist angles. In contrast, in cold-induced precipitates, a loss of anti-parallel beta sheet content is observed. This difference is partly explained by the differential effects of temperature on different non-covalent interactions stabilizing protein structures. The native beta sheet content of gamma B crystallin (approximately 50%) is raised in heat- (approximately 60%) and refolding-induced (approximately 58%) precipitates, but lowered in cold- (approximately 41%), and UV-induced (approximately 44%) precipitates. Cold precipitates also display approximately 26% helical content. All four aggregates have distinctively different morphological characteristics; this appears to be in keeping with their distinctively different secondary structural contents.
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http://dx.doi.org/10.2174/092986610791760414DOI Listing
September 2010

Metal-catalyzed proteolysis, conformational antigenicity, photosensitized oxidation, and electrical dysfunction explain the pathogenicity of protein aggregates.

Med Hypotheses 2010 Sep 8;75(3):294-8. Epub 2010 Apr 8.

Department of Immunopathology, Postgraduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh 160012, India.

It is widely accepted that protein aggregates tend to be pathologic, although little is known about why they are pathologic. Here, we summarize published findings about protein aggregates which have implications for pathology, but which have not yet been covered in any review or hypothesis on the subject, to the best of our knowledge. These findings suggest that protein aggregates can: (i) act as proteases, using exposed surface serines, (ii) function as immunogens, using novel conformational epitopes, (iii) behave as photosensitization-aids, using a novel peptide-based fluorescence, and (iv) act as electrical conductors, using electrons tunneling through hydrogen-bonded networks of peptide bonds. The potential pathological consequences of each finding are speculated upon.
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http://dx.doi.org/10.1016/j.mehy.2010.03.007DOI Listing
September 2010

Creation of a new eye lens crystallin (Gambeta) through structure-guided mutagenic grafting of the surface of betaB2 crystallin onto the hydrophobic core of gammaB crystallin.

FEBS J 2009 Jun 7;276(12):3341-53. Epub 2009 May 7.

Division of Protein Science & Engineering, Institute of Microbial Technology, Chandigarh 160036, Council of Scientific & Industrial Research, New Delhi, India.

The degree of conservation of three-dimensional folds in protein superfamilies is greater than that of amino acid sequences. Therefore, very different groups of residues (and schemes of residue packing) can be found displayed upon similar structural scaffolds. We have previously demonstrated the workability of a protein engineering-based method for rational mixing of the interior features of an all-beta enzyme with the substrate-binding and catalytic (surface) features of another enzyme whose sequence is not similar but which is structurally homologous to the first enzyme. Here, we extend this method to whole-protein surfaces and interiors. We show how two all-beta Greek key proteins, betaB2 crystallin and gammaB crystallin, can be recombined to produce a new protein through rational transplantation of the entire surface of betaB2 crystallin upon the structure of gammaB crystallin, without altering the latter's interior. This new protein, Gambeta, consists of 61 residues possessing the same identity at structurally equivalent positions in betaB2- and gammaB crystallin, 91 surface residues unique to betaB2 crystallin, and 27 interior residues unique to gammaB crystallin. Gambeta displays a mixture of the structural/biochemical characteristics, surface features and colligative properties of its progenitor crystallins. It also displays optical properties common to both progenitor crystallins (i.e. retention of transparency at high concentrations, as well as high refractivity). The folding of a protein with such a 'patchwork' residue ancestry suggests that interior/surface transplants involving all-beta proteins are a feasible engineering strategy.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07059.xDOI Listing
June 2009

Attenuation of ionic interactions profoundly lowers the kinetic thermal stability of Pyrococcus furiosus triosephosphate isomerase.

Biochim Biophys Acta 2009 Jun 21;1794(6):905-12. Epub 2009 Mar 21.

Institute of Microbial Technology, Council of Scientific and Industrial Research, New Delhi, India.

We investigate here the high structural stability of Pyrococcus furiosus triosephosphate isomerase (PfuTIM) by exploring the effects - upon the protein's structure and kinetic thermal stability - of modulation of its ionic interactions through pH variations, and mutations. PfuTIM shows comparable structural contents at pH 3.0, 7.0 and 10.0. However, at pH 3.0, subtle changes are seen in the protein's surface hydrophobicity and association status, and its kinetic thermal stability is profoundly reduced (as evidenced by its facile heat- and cold-mediated denaturation, characterized by a high degree of hysteresis and irreversibility). Increase in ionic strength through addition of salt counters the reduction of stability, and reversal of pH facilitates partial refolding. Further, a mutated form of PfuTIM (mPfuTIM) lacking 4 key charged residues involved in ionic interactions displays a structural content identical to PfuTIM but profound reduction in kinetic stability to thermal and chemical denaturation, as well as evidence of partial unfolding at temperatures between 90 degrees C and 100 degrees C, unlike PfuTIM. We conclude, therefore, that ionic interactions (which are known to determine protein thermodynamic stability) can also contribute significantly to protein kinetic thermal stability.
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http://dx.doi.org/10.1016/j.bbapap.2009.03.005DOI Listing
June 2009