Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins

Journal Pre-proof Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins Rajender Jena, Dushyant K Garg, Mohan Murali V Achary, Jasdeep Singh, Rachana Tomar, Lipsa Choudhury, Ruby Bansal, Bishwajit Kundu

PII:

S0168-1656(20)30019-5

DOI:

https://doi.org/10.1016/j.jbiotec.2020.01.017

Reference:

BIOTEC 8594

To appear in:

Journal of Biotechnology

Received Date:

8 November 2019

Revised Date:

27 January 2020

Accepted Date:

30 January 2020

Please cite this article as: Jena R, Garg DK, Achary MMV, Singh J, Tomar R, Choudhury L, Bansal R, Kundu B, Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins, Journal of Biotechnology (2020), doi: https://doi.org/10.1016/j.jbiotec.2020.01.017

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Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins

Rajender Jena1,£, Dushyant K Garg2£, Mohan Murali V Achary3, Jasdeep Singh1, Rachana Tomar1, Lipsa Choudhury4, Ruby Bansal5, Bishwajit Kundu1* [email protected]

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Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi,

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India

School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

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International Center of Genetic Engineering and Biotechnology, New Delhi, India

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School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi, India

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Department of Chemical Engineering Indian Institute of Technology Delhi, New Delhi,

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2

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India

Current Affiliation

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RJ: Serum Institute of India Private Limited, Pune, India

RT: Vanderbilt University, Nashville, Tennessee, United States £

These authors contributed equally

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*Corresponding author

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Highlights

An ATP-independent molecular chaperone as a potential protein stabilizer is

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proposed. 

NPfA imparts thermal stability and activity enhancement of substrate proteins.

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The mechanism relies on transient binding of NPfA to substrate proteins under stress and subsequent release upon removal of stress.

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NPfA thus could be utilized for various biotechnological applications.

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Abstract Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosus L-asparaginase, (NPfA, a protein chaperone

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lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG

peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed

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increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine,

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sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD

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polymerase (~ 40% increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and

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size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate

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protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is

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demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications. Abbreviations: NPfA, N-terminal domain of Pyrococcus furiosus L-Asparaginase; APX, ascorbate peroxidase enzyme; ARI-HAb, anti-rat IgG-Horse Radish peroxidase-linked

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antibody; AMI-HAb, anti-mice IgG- Horse Radish peroxidase-linked antibody; KOD, Thermococcus kodakarensis; PfCSP, Plasmodium falciparum circumsporozoite protein; OPD, o-phenylene diamine.

Keywords: L-Asparaginase; protein domain; molecular chaperone; activity enhancer; protein

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aggregation; osmolytes.

1. Introduction

The biological function of a protein is dictated by its correctly folded conformation. The

conformation of most of the proteins is labile and thus a slight change in physico-chemical

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environment may render them inactive (Colón et al., 2017). The susceptibility of proteins to

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various stress is a major impediment in the production, formulation, packaging, transportation and their day to day use in therapeutic and pharmaceutical practices. Inactivation during any

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of these processes cause significant economic loss (Romero-Romero et al., 2016). Several efforts have been made to enhance the activity and stability of proteins of biopharmaceutical

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importance. This includes the addition of osmotically active molecules such as amino acids, sugars and polyols, such as dextran, trehalose and sorbitol (Kaushik and Bhat, 2003; Sasahara

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et al., 2003; Rydeen et al., 2018). Such molecules act either by stabilizing the proteins or by inhibiting the aggregation. In some cases, fusion of proteins with stable partners has been

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carried out to obtain desired stability (Cordes et al., 2012; Qiu et al., 2010). Such approaches however suffer from enhanced workload of introduction of fusion step for each substrate protein. Chemical conjugation of moieties such as glycans, polyethylene glycol (PEG) and other hydrophilic molecules have been reported to enhance the shelf-life of some proteins. But these processes are tedious as each protein requires different sets of trial and error

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experiments before finding a suitable candidate for conjugation (Constantinou et al., 2012; Kolate et al., 2014; Liebner et al., 2014). A natural defensive mechanism of every organism against stressful environment is their ability to use factors known as chaperones to protect their cellular proteins (Bakthisaran et al., 2015). Small HSPs (sHSPs) are one class of such chaperone that displays considerable sequence similarity with vertebrate eye lens protein α-crystallin (α-C) (Ingolia and Craig, 1982; Jakob et al., 1993b; Nakamoto and Vigh, 2007). Proteins lacking the α-crystallin

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domain with sHSP-like properties are also reported (Librizzi et al., 2014) such as FK506 binding protein of Methanococcus thermolithotrophicus, aspartyl aminopeptidase (AAP) from S. pombe, nucleolar proteins, microtubule associated proteins, proteins involved in

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transcription like NusA and common proteins like serum albumin (Faircloth et al., 2009; Finn et al., 2012; Furutani et al., 2000; Lee et al., 2009; Li et al., 2013; Szebeni and Olson, 1999).

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In many cases, such proteins like DnaK, α-crystallin, casein etc. have been used as protein

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stabilizers (Katakam and Banga, 1997; King, 1997; Sabbaghian et al., 2011). The approach is easy, as it requires simple addition of suitable protein stabilizer into the solution which not only stabilize but also prevents the formation of amyloid fibers in substrate proteins. This

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becomes crucial for many protein formulations such as insulin which tend to undergo amyloidogenesis with prolong incubation (Librizzi et al., 2014).

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Previously in our lab N-terminal domain of L-asparaginase from Pyrococcus furiosus (NPfA)

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was identified as a novel chaperone which prevented variety of substrate proteins from thermal and refolding mediated aggregation (Tomar et al., 2013). In addition, it has also been reported to disaggregate preformed amyloid aggregates. NPfA has also been reported to confer thermo-tolerance to E. coli by rescuing a set of non-redundant cellular proteins, thus affirms its chaperoning potential (Jena et al., 2018). NPfA acts in an ATP-independent manner and is non-homologous to known ATP-independent small heat shock proteins. In the

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present study we tested the effect of NPfA on the biological activity of three model enzymes namely ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase from Thermococcus kodakarensis. The thermal protection provided by NPfA was comparably higher than the commonly used osmolytes of different classes, viz. methylamines, polyols and sugars. We report here that NPfA interacts with these biotechnologically important enzymes and not only enhances their stability against thermal

2. Materials and methods 2.1. Cloning, expression and purification of proteins

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denaturation, but also enhances their biological activity by several folds.

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For NPfA a previously developed recombinant plasmid was used as template to mutate the only tryptophan into phenylalanine (Tomar et al., 2013). A PCR based site directed

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mutagenesis was carried out using previously used primer pairs that were used for the

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mutation of N-terminal tryptophan (Garg et al., 2015). The mutated plasmid was propagated into E. coli (Top10) cells and subsequently transformed into E. coli BL21-DE3 Codon Plus

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expression strain. For the recombinant Pennisetum glaucum (Pg) APX protein expression, the coding region of PgAPX gene (GenBank: EF495352.1) was PCR amplified using cDNA as

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template and cloned into NdeI and NotI sites of pET-28a (+) vector. For both NPfA and APX, their recombinant clone was transformed into E. coli BL21-DE3 codon plus expression

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strain. The transformed cells were grown by incubating at 37 °C in Luria Bertani medium containing 50 µg ml-1 kanamycin. As the absorbance (A600) of the culture reached 0.6, isopropyl β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.2 mM and the culture was grown overnight at 25 °C. The expression level was analyzed by 15% SDS-PAGE. For purification, induced culture was lysed by sonication in lysis buffer (10 mM Tris-HCl, 100 mM NaH2PO4 and 5 mM imidazole pH 8.0) and the soluble NPfA domain was 6

purified through Ni–NTA chromatography according to the manufacturer’s instructions (Qiagen, Germany). Both the APX and NPfA purification protocols followed identical unit operations except for two changes. In the case of APX, IPTG was used at 1 mM final concentration and the culture was grown for 4 hours post induction at 37 °C. All the purification steps were performed at 4 °C. The anti-Rat IgG (whole molecule)-horseradish peroxidase antibody produced in rabbit (ARI-HAb) and anti-mouse IgG (whole molecule)horseradish peroxidase antibody produced in goat (AMI-HAb) was purchased from Sigma-

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Aldrich (USA) and KOD DNA polymerase, isolated from was purchased from Toyobo (Japan; cat no: KOD-201). PBS was purchased as tablets (Sigma Aldrich, USA), where one tablet dissolved in 200 mL of deionized water yields 0.01 M phosphate buffer, 0.0027 M

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potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C.

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a. In-gel Assay protocol for APX

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2.2. Characterization of purified NPfA and tryptophan mutant

For analyzing APX in-gel activity the purified protein was run on a native PAGE (10 %). 2

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mM ascorbic acid was added to the electrode buffer and the gel was pre-run for 30 min before the protein samples were loaded (Mohan Murali Achary et al., 2012). After

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electrophoresis, the gel was immersed for 30 min in 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM ascorbic acid with a change of the solution every 10 min. The gel was

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further soaked in 50 mM sodium phosphate buffer containing 4 mM ascorbic acid and 2 mM H2O2 for an additional 20 min before a brief wash in 50 mM sodium phosphate buffer. Finally, the gel was incubated in 50 mM sodium phosphate buffer (pH 7.8) containing 100 mM Tetramethylethylenediamine (TEMED) and 1 mM Nitro Blue Tetrazolium (NBT) until it turned uniformly blue except at positions corresponding to APX band. Upon achieving the best contrast of colorless APX bands the reaction was stopped by rinsing the gel in water.

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b. Circular dichroism spectroscopy All CD spectra were acquired using J-815 CD spectrophotometer (Jasco corporation, Japan), equipped with a peltier temperature controller. Protein (0.2 mg ml-1) in phosphate buffer saline (PBS) was taken and spectra were recorded in the wavelength range of 200-250 nm using a quartz cuvette of 1 mm pathlength (Starna, UK). The scanning speed, band width and data pitch were 50 nm min-1, 1 nm and 1 nm, respectively. Two scans for each measurement

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were averaged. For equilibrium unfolding experiment, 0.2 mg ml-1 protein was incubated with varying concentration of guanidine hydrochloride (GdnCl; Invitrogen, Carlsbad, USA) for 16 hours at 23 ℃. To achieve this, a 1 mg ml-1 protein stock was prepared in double

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distilled water and an 8 molar stock of GdnCl was prepared in 10 mM sodium phosphate buffer (pH 7.4). The protein was diluted in denaturing buffers containing 1x PBS and

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increasing concentrations of GdnCl (0-6M), such that the final protein concentration was 0.2

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mg ml-1 in each reaction mixture. The CD spectra of each sample was recorded and ellipticity values at 222 nm were plotted as a measure of protein unfolding. The percentage unfolding (FU%) was calculated using the following equation:

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FU% = [(Y-YN) ÷ (YD - YN)]

Where Y is the ellipticity value at 222 nm, and YN and YD are the ellipticity values at 222 nm

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of the native and completely denatured proteins, respectively.

2.3. Effect of NPfA and osmolytes on APX enzyme activity To measure APX activity, 15 nM APX was added to a reaction mixture containing 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid and 0.2 mM H2O2 (Chen and Asada, 1989). The hydrogen peroxide-dependent oxidation of ascorbate was followed by recording the decrease in absorbance at 290 nm (ε= 2.8 mM-1 cm-1) and the enzyme activity was expressed in µmoles of oxidized ascorbate mg-1 protein min-1. 8

To study the effect of increasing NPfA concentrations on APX activity, 2.5 µM aliquots of purified recombinant APX were mixed with NPfA at molar ratios of APX:NPfA of 1:1, 1:2, 1:5, 1: 10 and 1: 20 and incubated at 40 °C for 10 min. Control experiments were carried out using BSA in place of NPfA. In a subsequent experiment, the chaperoning effect was checked at different temperatures ranging from 30 to 45 °C keeping the molar ratio of NPfA:APX at 1:2 (obtained from aforementioned experiment) after incubation for 10 min. To compare the benefits of NPfA with those of different classes of osmolytes on APX activity; 1.5 molar stocks of glycine betaine, sorbitol and trehalose were prepared in 50 mM sodium phosphate (pH 7.0). APX was incubated for 10 minutes at 40 °C in 0.5 M

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and 1.0 M concentrations of osmolytes. The activity measurement of resultant mixture was carried out as mentioned above.

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2.4. Effect of NPfA on the activity of I-HAbs

ARI-HAb and AMI-HAb (833 nM) were heated in the absence or presence of 22 µM NPfA at

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temperatures ranging from 30 to 70 °C for 60 minutes and analyzed by indirect enzyme linked immunosorbant assay (ELISA). Microtiter plates (96 wells) were coated in triplicates (18 h, 4 °C)

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with 1 μg ml-1 (0.1 μg in each well) of Plasmodium falciparum circumsporozoite protein (PfCSP) in 100 μl coating buffer (0.05 M Na2CO3, 0.05 M NaHCO3, pH 9.6). The unbound antigen was washed

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twice with the coating buffer containing 0.1% Tween-20. The wells were blocked with 2 % bovine serum albumin (BSA) in phosphate buffered saline (PBS; pH 7.4) for 2 h at 37 °C. For standard

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curve, wells in triplicates were coated with titrating amount of PfCSP that was obtained through serial dilution of 3 µg ml-1 of PfCSP to a final concentration of 0.012 µg ml-1. The immobilized antigen was

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incubated with primary antibody (PfCSP-specific rat antisera or mice antisera) (100 μl/well) in triplicates at 1:20000 dilution in Tris borate saline containing 0.1% Tween-20 (TBS-T) followed by incubation for 2 h at room temperature, followed by washing three times with TBS-T. In the subsequent step, secondary antibody ARI-HAb or AMI-HAb that was heat-treated in the presence of NPfA was diluted in blocking buffer (1:20,000) and 100 μl of this mixture was added to wells. The plate was washed three times with TBS-T. Heat treated ARI-HAb or AMI-HAb in absence of NPfA

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was used as a control. The plate was further incubated for 1 h at room temperature. 50 μl of chromogenic substrate o-phenylenediamine (OPD) in sodium citrate buffer (pH 5.0) was added and incubated for 30 min. The reaction was stopped with 100 μl H2SO4 (2 N) and absorbance at 492 nm was measured using Versamax ELISA reader.

2.5. Effect of NPfA on KOD DNA polymerase activity The effect of NPfA on KOD DNA polymerase activity was evaluated by PCR amplification of standard DNA template of 800 bp length. PCR reactions of 50 µl final volume were prepared by

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mixing 5 µl of 10X KOD polymerase buffer, 1 µl dNTP (10 mM), 1 µl each of forward and reverse primers (150 ng µl-1), and 1.5 µl of DNA template (equivalent to 20 ng). 1U µl-1 KOD polymerase

stock was used at final concentrations of 0.01, 0.005, 0.0037 and 0.0025 U µl-1 in the PCR reactions.

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NPfA was added to each PCR reaction at a final concentration of 0.2 µg µl-1 (9 µM). 2 control reactions were maintained where NPfA was substituted either with KOD buffer or with BSA.

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Standard PCR conditions were followed and products were analyzed on 1% agarose gel and quantified with densitometry.

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To assess the effect of NPfA on the thermal stabilization of KOD DNA polymerase separate set of experiments were performed. KOD polymerase at a final concentration of 0.005U µl-1 in 20 mM Tris-

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HCl (pH 7.5) either alone or in the presence of 0.2 µg µl-1 (9 µM) NPfA was taken in a final reaction volume of 20 µl and heated at 97 °C for 10, 20 30 and 40 minutes. After heat treatment remaining

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components of PCR mixture (template, dNTPs, buffer, primers etc.) were added to make up the final

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volume to 50 µl. The residual activity was analyzed as mentioned above.

2.6. Effect of NPfA on heat induced changes in APX All fluorescence studies were performed on a Varian Cary Eclipse spectrofluorimeter (Agilent), attached with a water circulatory bath for temperature adjustment. The concentration of APX and NPfA were 21.4 and 45 µM, respectively. Thus a 1:2 molar ratio was maintained. The structural changes in APX at varying temperature or time were monitored at 340 nm using excitation

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wavelength at 295 nm. The excitation and emission slit widths were 2.5 and 5 nm, respectively. The scattering experiments were carried out by setting excitation and emission wavelength at 400 nm, respectively. The percentage signal changes were calculated assuming initial signal as 100%. To compare this with those of osmolytes APX was incubated at two different concentrations of glycine betaine, sorbitol and trehalose at 40 ℃ for 30 minutes. The samples were cooled down to 23 ℃, and intrinsic tryptophan fluorescence was measured as mentioned above. The intensity at 340 nm for the unheated sample was considered as 100% structured. Deviation in intensity was plotted as % change

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in structure.

NPfA-APX interaction studies

2.7.1. Isothermal titration calorimetry

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Isothermal titration calorimetry (ITC) was performed using Micro Cal-iTC 200 (Amherst,

MA, USA) where 14µM APX was titrated against 45µM of NPfA (both in PBS, pH 7.4). In

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all experiments, 1µL of NPfA was injected into APX solution with a pre-injection delay of

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150s for complete equilibration. Titrations were carried out at both 25 °C and 40 °C with a stirring speed of 400 rpm. The heat evolved or absorbed in the process was measured. Control

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experiments were performed where NPfA was titrated into PBS alone to account for the heat of dilution. The data was analyzed using Origin v. 7.0 software and fitted with two site

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sequential binding model to obtain thermodynamic parameters. The binding constants K1 and K2 were defined relative to the progress of saturation, so that [𝑀𝑋 ]

[𝑀𝑋 ]

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1 2 𝐾1 = [𝑀][𝑋] 𝐾2 = [𝑀𝑋][𝑋]

Where M is the free concentration of the macromolecule and X is free concentration of ligand.

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2.7.2. Size-exclusion chromatography Size-exclusion chromatography was performed using Superdex 200 10/300 column fitted to an AKTA purifier FPLC system (GE Healthcare, Uppsala, Sweden). The column was calibrated by using standard molecular weight (Mr) marker proteins. The void volume was determined using dextran 2000. A curve between Kav and log Mr, was plotted, where Kav = (Ve - Vo)/(Vc - Vo). Here Ve = elution volume, Vc = geometric column volume and Vo = void volume. Prior to sample analysis, the column was equilibrated with 3 column volume of PBS. The flow rate of elution was 0.5 ml min-1. The NPfA APX mixture was pre-heated at 40 ℃ before injecting into the

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column. As a control, APX and NPfA were analyzed separately. The data were acquired and analyzed using UNICORN software.

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2.7.3. Dynamic Light Scattering

Dynamic light scattering (DLS) was performed using a miniDAWN Tristar laser photometer

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with a quasi-elastic light scattering (QELS) attachment (Wyatt Technology Corp. Santa

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Barbara, CA), equipped with 664 nm laser. The data were collected at 90º scattering angle and analyzed using inbuilt Astra software. Protein samples individually or as a mixture

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containing 0.5 mg ml-1 APX and 1.0 mg ml-1 NPfA (80 µL each) were taken in a quartz cuvette and diffusion coefficient (DT) was determined. Before data acquisition, the protein was filtered through 0.22µ syringe filter followed by degassing and centrifugation at 12,000

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g. Each protein was subjected to thermal stress at 40 ℃ for 10 minutes before data

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acquisition. Protein mixture was formed with 1:2 (APX: NPfA) molar concentration. Subsequently, the protein solutions were cooled down to 25 ℃ and data was collected. Hydrodynamic radii in nm (Rh) was calculated from values of DT using Stokes-Einstein equation, Rh = kbT/6πηDT

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Where kb is Boltzmann's constant, T is temperature, DT is translational diffusion coefficient and η is the solvent viscosity.

2.8.

Statistical analysis

The biochemical experiments were conducted in three independent technical replications and repeated twice. The mean values ± standard errors (SE) are presented in the Figures. For ELISA data, all statistical measures were conducted in Prism (GraphPad software, v 6.0). Data were tested for

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statistical significance using paired t-test to compare two and multiple experimental groups. For all statistical tests, p < 0.05 was considered as significant.

2.9. APX-NPfA complex formation

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To assess the type of complex formed between NPfA and APX, ZDOCK webserver was

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employed (Pierce et al., 2013). For docking of two NPfA (PDB id: 4ra6) molecules with APX enzyme (PDB id: 2xif), ZDOCK, a fast Fourier transform (FFT)-based, initial-stage

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rigid-body molecular docking algorithm was used. This had previously achieved good performances in the CAPRI challenge for predicting protein–protein complexes. Taking into

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account the biological assembly of APX and the interfacial residues of both proteins, the resulting hits with highest binding score were chosen. Hetero-trimer model which displayed

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highest number of multiple inter-protein interactions was further subjected to molecular

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dynamics simulations.

2.10.

MD simulations

Molecular dynamics simulations of 100 ns for both APX alone and in complex with NPfA (APX: NPfA :: 1:2) were done using GROMACS 5.1 using the CHARMM36 all-atom force field (Huang and MacKerell, 2013; Makarewicz and Kazmierkiewicz, 2013; Makarewicz and Kaźmierkiewicz, 2016). The periodic box was solvated using SPC water model and counter 13

ions were added to maintain electro-neutrality. The system was then minimized for 50000 steps using a steepest decent algorithm followed by an equilibration run of 100 ps in NVT ensemble with restrains on the protein atoms. The NPT ensemble was used for production simulation. Systems were simulated at 330K, maintained by a Berendsen thermostat with a time constant of 1 ps. Pressure coupling was done employing a Parrinello-Rahman barostat using a 1 bar reference pressure and a time constant of 2.0 ps with compressibility of 4.5e-5 bar using isotropic scaling scheme. Electrostatic interactions were treated using the Particle

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Mesh Ewald method. Trajectory analysis was done using in-built GROMACS tools. Images were created using PyMol and Discovery Studio Visualizer 4.5 (Dassault Systèmes BIOVIA;

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The PyMOL Molecular Graphics System).

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3. Results

3.1. Expression, purification and characterization of recombinant proteins The recombinant NPfA and APX were over-expressed in E. coli (DE3 codon plus) expression system

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separately. Majority of the recombinant proteins were partitioned into the soluble fraction. Proteins were purified using immobilized Ni–NTA affinity chromatography and confirmed by western blot or

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in-gel assay (Fig. 1A and 1B). The site directed mutagenesis was further confirmed by measuring the tryptophan emission fluorescence spectra of WT and mutant NPfA (W60F). At 295 nm excitation

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wavelength tryptophan residue is selectively excited. Minimal spectral intensity of the mutant confirmed the absence of Trp residue (Fig. 1C). The CD signature of the WT and W60F were overlapping, indicating that mutation had no effect on the secondary structure of NPfA (Fig 1D). GdnCl mediated denaturation profiles of WT and W60F were also co-incident (Fig 1E), which proved that the stability of both the proteins were same. We further examined the chaperoning activity of W60F by observing its ability to thermally protect the substrate protein (APX) and found it to be 14

comparable with that of WT (Fig 1F). Thus, it was evident that W60F mutation had no effect on the structure and function of NPfA.

3.2. Effect of NPfA on APX activity Standard APX activity was measured at 40 °C (pH 7.2) which was found to be its optimum temperature (Fig 2B). The activity of APX without NPfA was considered as 100 %. Increasing concentrations of NPfA showed profound effect on APX activity as compared to the control (in absence of NPfA or in the presence of BSA). Although at all molar ratios of APX:NPfA increment in APX activity was observed, (except for 1:20) but most significant enhancement (78 %) of was

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recorded at 1:2 ratio (Fig. 2A). Keeping the concentration of APX:NPfA at 1:2, we subsequently analyzed the effect at different temperatures ranging from 30 to 45 °C (Fig. 2B). Regardless of

variations in temperature, presence of NPfA always resulted in enhancement of APX activity, which

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was found to be maximum at the enzyme’s optimum temperature (40 °C, Fig. 2B).

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3.3. Comparison of NPfA with commonly used osmolytes in thermoprotection of APX To further compare the effect of NPfA with commonly used osmolytes on APX, we incubated APX at 40 °C for extended time (30 minutes) in the presence of glycine betaine, sorbitol and trehalose. Two

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commonly used concentrations, 0.5 M and 1 M, of osmolytes were used in this study. After cooling from 40 °C, the activity and fluorescence intensity (representing structural stability) of APX reduced

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to ~ 63% compared to unheated control (Fig. 3 A and 3B). In contrast, presence of NPfA conferred resistance and maintained residual activity and fluorescence signal of ~83%, signifying its protective

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role. Osmolytes showed either similar or increased residual activities or fluorescence intensities, representing comparable or lower protective role as compared to that conferred by NPfA. We observed consistent structure-function relationship with APX, as discernible from Fig. 3C, where fluorescence and activity profiles overlapped. We thus conclude that NPfA appreciably rescued the structure (fluorescence profile) and concomitantly the activity of APX.

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3.4. Effect of NPfA on I-HAbs activity To examine whether NPfA can protect the ARI-HAb and AMI-HAb from thermal stress both were heat treated in the absence and presence of NPfA at different temperatures ranging from 30 to 70 °C for 60 minutes (Fig. 4). These samples were used as secondary antibody to quantitate PfCSP which was already captured by a primary antibody produced either in rat (for ARI-HAb) or in mouse (for AMI-HAb) (as mentioned in the material and methods section). The absorbance values were converted to percentage activity considering 100% activity for the untreated control at 30 °C. Heat treated ARI-HAb and AMI-HAb in the absence of NPfA displayed a decreasing trend in activity with

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increase in temperature. In contrast, incubation of ARI-HAb and AMI-HAb with NPfA resulted in

significant and consistent increase in the absorbance values. Beyond 60 °C however, NPfA failed to exhibit its protective function.

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3.5. Activity enhancement of DNA KOD polymerase

To further assess activity enhancement effect of NPfA, a fixed amount of NPfA (0.2 mg ml-1) was

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added to PCR mixtures containing decreasing concentrations of KOD DNA polymerase (0.01, 0.005,

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0.0037 and 0.0025 U µl-1). In control reactions, the NPfA was either omitted or replaced with 0.2 mg ml-1 BSA. Amplification of DNA template using these mixtures were analyzed by 1% agarose gel electrophoresis and quantitated through densitometry. It was found that amplification was prominent

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in all the reaction catalyzed through NPfA protected KOD polymerase as compared to the corresponding controls (Fig. 5A). The BSA containing samples also did not show amplification below

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0.005 U µl-1 concentrations, confirming that the enhancement effect is specifically imparted by NPfA. Interestingly, comparison of respective controls and NPfA containing test samples displayed

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considerable percentage increase in band intensities at lower KOD polymerase concentrations (8% and 21% increase respectively, at 0.01 U µl-1 and 0.005 U µl-1 of KOD polymerase). Further lowering of KOD polymerase concentration (0.0037 and 0.0025 U µl-1) in control reaction did not yield any amplification whereas the corresponding NPfA containing sample showed a clear band. In a separate set of reactions 0.005U µl-1 of KOD polymerase was pre-heated to 97 °C (with or without 0.2 µg µl-1 of NPfA) for varying time periods and analyzed on 1% agarose gel as above. It

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was observed that samples heated in the absence of NPfA yielded bands whose intensity gradually decreased with time up to 20 minutes of incubation (Fig. 5B). Heating beyond this time resulted in a complete loss of KOD polymerase activity as they failed to show any band on agarose gel. In contrast, presence of NPfA in the reaction mixture resulted in a robust band signal for all incubation time periods. BSA at 0.2 µg µl-1 was used as a control. Densitometry results suggest that in the control experiments where no pre-heating was done, addition of NPfA resulted in 1.14 times higher band intensities as compared to the control. Band comparison showed further intensification (~2.79 times) for 20 minutes heated samples in presence of NPfA. The above results suggest that NPfA act as

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thermo-stabilizer and help the enzyme to retain its activity for longer duration at high temperatures.

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3.6. NPfA effectively protects the heat induced aggregation of APX by protecting its structure.

To understand the effect of temperature on the structure of APX, its fluorescence intensity at 340 nm

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after exciting at 295 nm was measured with at different temperatures (Fig. 6A). The absence of tryptophan residue in NPfA enabled us to monitor fluorescence signal of APX exclusively, even in the

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presence of NPfA. It was observed that after heating at varying temperature for 10 minutes and subsequent cooling to 25 °C, APX regained original signal up to 30 °C. It loses significant signal

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irreversibly at 35 °C. Interestingly, a slight enhancement in the signal intensity was observed beyond 35 °C which eventually declined at 50 °C. The same trend could be observed in the activity profile of APX (Fig. 2B). It might be possible that at low denaturation stress (thermal and chemical), when

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protein is partially unfolded, the refolding might not be efficient. At higher denaturation stress the

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protein tend to lose most of the structure and refold back efficiently. This may be due to the fact that the partially unfolded states of proteins harbor active hydrophobic patches and are therefore more prone towards aggregation. The signal loss for APX was significantly less in the presence of NPfA, validating the protective effect of NPfA. To further understand the structural loss with respect to temperature, APX was subjected to a continuous temperature scan and intensity at 340 nm was recorded as a measure of structural integrity (Fig. 6B). There was a continuous structural loss in APX in the presence or absence of NPfA. However, 44 °C and beyond, NPfA appeared to reduce the loss in 17

the structure compared to APX alone, which continuously lost structure up to 65 °C. To study the time dependent aggregation behavior of APX, scattering was monitored at 40 °C (Fig. 6C). The aggregation profile of APX was significantly high compared to its aggregation profile in the presence of NPfA. The aggregation of NPfA was negligible in the employed regime. The time dependent structural change was monitored through fluorescence intensity at 40 °C and it was found that in the presence of NPfA, the structural loss in APX was 30% which was significantly lower in the case of APX alone (55%; Fig. 6D).

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3.7.NPfA-APX interaction studies 3.7.1. Isothermal titration calorimetry

ITC was carried out to determine stoichiometry and thermodynamics of APX-NPfA binding

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at 25 °C and 40 °C. The latter temperature was chosen as APX displayed maximum activity

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at this temperature in presence of NPfA (Fig. 7A and 7B). The ITC profiles at 25 °C and 40 °C indicated an exothermic and endothermic nature of interaction respectively, based on heat

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of evolution in both systems. The data fitted well with two-site sequential binding model and the thermodynamic parameters obtained are summarized in Table 1. The analysis showed

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that NPfA-APX interaction at two binding sites were differently governed by both entropic and enthalpic contributions with a binding stoichiometry of 2:1 (NPfA:APX) (Table 1).

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Interestingly at 25 °C, we observed 10 fold reduction in association of NPfA-APX interaction as compared to that of 40 °C (K1 and K2, Table 1). The ITC outcomes were further

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supported by simulation outcomes (described later).

3.7.2. Size-exclusion chromatography The ITC data at 25 °C displayed weak binding between NPfA and APX. This could be a result of momentary association between transiently populated unfolded species and NPfA. Size exclusion chromatography corroborated the same. The NPfA-APX mixtures were heated 18

at 40 °C and cooled down to 25 °C before subjecting to SEC (Fig. 7C). Two independent peaks, one each of NPfA and APX, were observed indicating non-existence of any stable interaction between the two proteins. Thus, it can be concluded that the NPfA-APX interaction which happened at 40 °C (as observed through ITC) was weakened as the temperature was lowered to 25 °C, resulting in two separate peaks.

3.7.3. Dynamic light scattering

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The size exclusion chromatography, led us to understand that NPfA and APX did not form stable complex at 25 ℃. However, to confirm that the proteins indeed interacted at 40 ℃,

dynamic light scattering experiments were performed (Fig. 8). We monitored temperature

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dependent changes in the size distribution patterns of the proteins and their reversibility to

native size when brought back from higher temperature. At 25 ℃, NPfA showed population

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of species around 4.54 nm (Fig. 8A). At 40 ℃, an additional oligomeric population of mean

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size 168 nm was observed (Fig. 8B), which when cooled to 25 ℃, almost regained its native size distribution (Fig. 8C). The mean size distribution of APX was 4.18 nm at 25 ℃ (Fig. 8D), which got shifted at 8.37 nm when heated at 40 ℃ (Fig. 8E). When cooled down to 25 ℃, the

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APX failed to regain its native size distribution (Fig. 8F), possibly because of the formation of aggregates. The NPfA-APX mixture at 25 ℃ (Fig. 8G) displayed mean size around 4.66

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nm which was comparable to individual NPfA or APX (Fig. 8A and 8D). This observation

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hinted that NPfA and APX did not form a stable complex at this temperature. When heated at 40 ℃, the NPfA-APX mixture partitioned into two major species of 6.46 nm and 179 nm (Fig. 8H). The size of smaller size species was larger than individual NPfA or APX, indicative of interaction between the two. The larger size species could be oligomerized NPfA as was the case with NPfA alone. When cooled down to 25 ℃, the NPfA-APX mixture regained its original size distribution (Fig. 8I). It is noteworthy that in this case, APX alone

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failed to regain its native size distribution (Fig. 8F). Thus we could conclude that at elevated temperature, NPfA significantly ameliorates the aggregation of APX by forming a complex and dissociates when the temperature stress is removed.

3.8. Protein-protein docking and molecular interaction insights through MD simulation The NPfA and APX protein-protein docking performed using ZDOCK were ranked on the basis of final score obtained from combination of shape complementarity, electrostatics and

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statistical potential. The 2:1 (NPfA:APX) structure was derived from sequential binding analysis of ITC data (Fig 7, Table 1). The top five models obtained showing tertiary

alignment of NPfA-APX complex have been shown in Supplementary Figure S1. To

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understand the molecular mechanism of the activity enhancement effects of NPfA, MD

simulations were performed for APX alone and modeled hetero-assembly at both 300 K and

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314 K (where highest activity was observed at 2:1 NPfA:APX complex, Fig. 9A). Backbone

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root mean square deviation (RMSD) plots for APX alone and in the presence of NPfA (1:2) equilibrated near 60 ns for all simulation runs (Fig. 10A). As expected, an increase in temperature of the systems led to thermal denaturation of APX alone which was inferred

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from increase in backbone RMSD and gyration radius analysis (Fig. 10A and B). Contrastingly at 314 K, APX in complex with NPfA unexpectedly showed minimal backbone

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deviations, corresponding to RMS deviations of APX alone at 300 K (Fig. 10A). Moreover,

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this complexation protected against temperature mediated unfolding of APX. The gyration radius of complexed APX remained essentially low compared to APX alone at 314 K indicating conservation of overall fold architecture. The ascorbic acid hydrolysis is regulated through crucial Lys 30, Cys 32 and Arg 172 at its binding site in APX (Fig.9B) (Sharp et al., 2004). Interestingly, in our simulation studies, the solvent accessibility of the ascorbic acid binding pocket of NPfA bound APX at 314 K was substantially higher (by ~ 1.5 nm2)

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compared to APX alone (at 300 K and 314) and APX-NPfA complex (at 314 K) (Fig. 10C). This could be attributed to increased interaction between NPfA and APX at specific temperature of 314 K which might have resulted in structural remodeling of active site. Thus we further checked H-bond occupancy over the entire simulation period for APX-NPfA complex at both 300 K and 314 K. The probability distributions for total number of H-bonds between APX and NPfA clearly showed shift in H-bond network at 314 K to higher number

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(~ 10 H-bonds) compared to system at 300 K (~ 5 H-bonds) (Fig. 10D).

Discussion

Enhancement of enzymatic activity is often desired for industrial and therapeutic

applications. To achieve this, majorly explored methods include immobilization, chemical

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modification, nano-encapsulation and other confinements (Gornowich and Blanchard, 2012;

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Zhang et al., 2015; Zhao et al., 2016). Application of small molecular chaperones in the case of industrially and therapeutically important enzymes has remained confined to the

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prevention of protein aggregation under stress conditions. In this report we show that a previously discovered small protein domain NPfA, not only acts as a chaperone but also

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increases biological activity of substrate proteins. In our study we employed model proteins from three diverse groups (plant proteins, antibodies and enzymes) namely ascorbate

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peroxidase (APX), IgG peroxidase antibodies and KOD DNA polymerase. In all the three cases, addition of NPfA, which has been established as a thermostable protein chaperone,

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resulted in several fold increment in activity. Generally for increasing stability, additives like inorganic salts, polyols, complex chemicals etc. are used. Interestingly, in our case none of these but a simple addition of NPfA in the standard assay buffer resulted in apparent enhancement in activity of the substrate proteins. sHSP are known to prevent denatured proteins from aggregating but are unable to refold nonnative proteins (Chang et al., 1996; Laksanalamai et al., 2001). However, instances of target 21

protein reactivation by sHSPs have been explained through their interference in the kinetic partitioning of substrate proteins between productive folding and aggregation states. Even at room temperature, owing to low unfolding free energy (5-15 kcal mol-1), protein molecules transiently sample unfolded state, which predisposes it towards proteolysis and reduced activity. It may be possible that addition of chaperone stabilizes the native state either by shifting the structural equilibrium towards folded state or by enhancing the kinetic stability of the native state (Garg and Kundu, 2017). In our case, NPfA successfully prevents thermal

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denaturation/aggregation of the target proteins at elevated temperatures (Fig. 2-5). Interestingly however, in case of all the 3 variety of proteins, an increment in the activity was also observed at low or optimum temperature. This could be case specific and may be

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reasoned as below.

Ascorbate peroxidase (APX) plays an important role in the metabolism of H2O2 in higher

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plants and eukaryotic algae thereby providing potential defense against oxidative damage

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caused by environmental stresses (Shigeoka et al., 2002). It is a heme containing enzyme with optimum activity at 40 °C. When NPfA was added in the assay system its activity almost doubled (Fig. 2) with no significant enhancement in the presence of BSA. The

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enhancement in activity may be due to the association of NPfA with APX active site pocket

catalysis.

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in a fashion that orients its substrate ascorbate to an ideal conformation facilitating easy

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Simulation estimates, ITC, DLS and SEC experiments supported our hypothesis on NPfA complexed-APX and thus temperature mediated activity of the complex. Through fluorescence measurement we inferred that NPfA prevented temperature mediated unfolding of APX (at 314 K) which could have resulted in retention of enzyme activity (Fig. 2B and 6A). This stability could be because of increased H-bond interactions and conformational changes at APX-NPfA binding interface which might have led to the stability enhancement.

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Furthermore, these temperature specific favorable interactions led to higher solvent accessibility at APX active site (Fig. 10C). We anticipate that the increased accessibility could accommodate the substrate (ascorbic Acid) in a better alignment and also facilitate easy dissociation of hydrolyzed products from the active site. It may be argued that such increase in solvent accessibility could negatively regulate substrate binding at active site. Such a case is likely to occur for highly unfolded architectures. In our case we observed only marginal increase in accessibility at active site along with specific molecular recognitions dominating

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over non-specific interactions. The addition of commonly used osmolytes enhanced the overall stability of APX, the extent of which was comparable or even lower than that conferred by NPfA. A general view on the

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mechanism behind protective effect of stabilizing osmolytes is their preferential exclusion from the protein backbone, which favour folded state of the protein (Ahmed et al., 2016;

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Rydeen et al., 2018). The NPfA however, exerts its protective effect through a transient

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interaction with the partially folded state, which in turn disfavor the intermolecular aggregation of the substrate proteins. This difference in the protective mechanism of action among NPfA and osmolytes thus explains the higher protective role of the former.

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Under stress condition, sHSPs are known to have “holdase” like properties that prevent the formation of irreversible aggregates by binding to their native forms in an ATP independent

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manner (Horwitz, 1992). It has also been reported that the refolding kinetics of substrate

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proteins vary in presence or absence of sHSPs. The protective effect of the substrate proteins in presence of sHSPs takes more time due to binding and release of the non-native structure with the sHSPs (Jakob et al., 1993a). In our case however, the protective effect of the NPfA was found to be instantaneous. Both ITC, DLS and SEC outcomes depicted weakening of interactions at lower temperature (25 ℃). This also points to a reversible transient binding event of NPfA with APX. The proposed mechanism however may be specific to APX and

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may vary in case of other substrate proteins. Both ITC and SEC data pointed towards a transient interaction at 25 ℃ which indicates that NPfA enhances the activity of APX by exerting its effect in non-associated state. In case of antibodies the recommended storage temperature is -20 °C or below owing to their susceptibility to thermal stress. Increasing their stability at higher temperatures is always desirable for increasing their shelf life. The 20-50 % increase in ARI-HAb and AMI-HAb activity seen in our case could be due to either one of the reasons: 1) there could be increase

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in the activity of Horse Radish Peroxidase (HRP). HRP is again a peroxidase which is used as a covalent tag for colorimetric detection of antibody function. Here the activity enhancement could be due to reasons similar to those mentioned for APX. 2) NPfA protects ARI-HAb and

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AMI-HAb against temperature denaturation and therefore acts a genuine chaperone as

reflected by increase in activity in the final read out. This explanation is most appealing as

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the increment in activity is more at higher temperatures (60 °C, Fig. 4) where substantial loss

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of activity is registered for non-NPfA containing antibody samples. However, a small increment in activity at 30 °C is also noticeable and could be because of NPfA providing a passive support for spontaneous refolding of some of the non-functional antibodies. The

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same reason could be attributed to the two fold higher activity of KOD DNA polymerase after prolonged heat treatment. KOD polymerase by itself is derived from thermophilic

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organism and therefore is likely to have natural resistance to thermal

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denaturation/degradation. Although its optimum temperature of activity is at 72 °C, it is advised to store KOD polymerase at low temperatures, preferably at -20 °C. Having established that NPfA has a thermo-protective function we sought to find out whether it imparts any beneficial function to KOD polymerase activity. We report that NPfA protected KOD polymerase from heat denaturation for a substantial period of time allowing the later to

24

undergo spontaneous refolding. In a way NPfA is similar in function to sHSP of Pyrococcus furiosus reported earlier (Chen et al., 2006; Laksanalamai et al., 2006). NPfA do not require ATP hydrolysis, but has been able to function as an efficient energy independent chaperone without interfering with the activities of the substrate proteins. From our study we propose that NPfA may be used as a crucial additive in enzyme formulations for enhancing their shelf life and biological activity, thus making the final formulations more

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stable and cost effective.

Credit Author statement

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This is to state that the authors of this manuscript contributed in the following way

Rajender Jena and Dushaynt K Garg- Experimentation, Methododlogy, Data analysis,

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Writing

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Jasdeep Singh- Computational and in silico work.

Mohan Murali V Achary and Rachana Tomar- activity assays and molecular biology work Lipsa Choudhury- ELISA and related work

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Ruby Bansal- Worked on generating ITC data

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Bishwajit Kundu- Conceptualization, analysis and writing

4. Conflict of interest Authors declare no conflict of interest

5. Acknowledgement

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RJ, JS and BK acknowledge the infrastructural support of IIT Delhi. Dr. Paushali Mukherjee is acknowledged for her help in statistical analysis of ELISA results. Authors thank IITD HPC facility for computational resources. The authors would also like to acknowledge funding support from the DBT Centre of Excellence for Biopharmaceutical Technology

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(number BT/COE/34/SP15097/2015).

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References 1. Ahmed, M., Namboodiri, V., Mathi, P., Singh, A.K., and Mondal, J.A. (2016). How Osmolyte and Denaturant Affect Water at the Air–Water Interface and in Bulk: A Heterodyne-Detected Vibrational Sum Frequency Generation (HD-VSFG) and Hydration Shell Spectroscopic Study. The Journal of Physical Chemistry C 120, 10252-10260. 2. Bakthisaran, R., Tangirala, R., Rao Ch, M., (2015) Small heat shock proteins: Role in cellular functions and pathology. Biochimica et biophysica acta 1854, 291-319.

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3. Chang, Z., Primm, T.P., Jakana, J., Lee, I.H., Serysheva, I., Chiu, W., Gilbert, H.F., Quiocho, F.A., (1996) Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. The Journal of biological chemistry 271, 7218-7223. 4. Chen, G.-X., Asada, K., (1989) Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties. Plant and Cell Physiology 30, 987-998.

-p

5. Chen, H., Chu, Z., Zhang, Y., Yang, S., (2006) Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus. Biotechnology letters 28, 1089-1094.

re

6. Colón, W., Church, J., Sen, J., Thibeault, J., Trasatti, H., and Xia, K. (2017). Biological Roles of Protein Kinetic Stability. Biochemistry 56, 6179-6186.

lP

7. Constantinou, A., Chen, C., Deonarain, M.P., (2012) Polysialic Acid and Polysialylation to Modulate Antibody Pharmacokinetics. Therapeutic Proteins. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 95-115.

na

8. Cordes, A.A., Carpenter, J.F., Randolph, T.W., (2012) Selective Domain Stabilization as a Strategy to Reduce Human Serum Albumin–Human Granulocyte Colony Stimulating Factor Aggregation Rate. Journal of Pharmaceutical Sciences 101, 2009-2016.

ur

9. Dassault Systèmes BIOVIA, D.S.M.E., Release 2017, San Diego: Dassault Systèmes, 2016.

Jo

10. Faircloth, L.M., Churchill, P.F., Caldwell, G.A., Caldwell, K.A., (2009) The microtubuleassociated protein, NUD-1, exhibits chaperone activity in vitro. Cell stress & chaperones 14, 95-103. 11. Finn, T.E., Nunez, A.C., Sunde, M., Easterbrook-Smith, S.B., (2012) Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands. The Journal of biological chemistry 287, 21530-21540. 12. Furutani, M., Ideno, A., Iida, T., Maruyama, T., (2000) FK506 binding protein from a thermophilic archaeon, Methanococcus thermolithotrophicus, has chaperone-like activity in vitro. Biochemistry 39, 453-462. 13. Garg, D.K., Kundu, B., (2017) Hyperthermophilic l-asparaginase bypasses monomeric intermediates during folding to retain cooperativity and avoid amyloid assembly. Archives of Biochemistry and Biophysics 622, 36-46. 27

14. Garg, D.K., Tomar, R., Dhoke, R.R., Srivastava, A., Kundu, B., (2015) Domains of Pyrococcus furiosusl-asparaginase fold sequentially and assemble through strong intersubunit associative forces. Extremophiles 19, 681-691. 15. Gornowich, D.B., Blanchard, G.J., (2012) Enhancement of Enzyme Activity by Confinement in an Inverse Opal Structure. The Journal of Physical Chemistry C 116, 12165-12171. 16. Horwitz, J., (1992) Alpha-crystallin can function as a molecular chaperone. Proceedings of the National Academy of Sciences 89, 10449-10453. 17. Huang, J., MacKerell, A.D., Jr., (2013) CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. Journal of computational chemistry 34, 21352145.

ro of

18. Ingolia, T.D., Craig, E.A., (1982) Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin. Proceedings of the National Academy of Sciences of the United States of America 79, 2360-2364. 19. Jakob, U., Gaestel, M., Engel, K., Buchner, J., (1993a) Small heat shock proteins are molecular chaperones. Journal of Biological Chemistry 268, 1517-1520.

-p

20. Jakob, U., Gaestel, M., Engel, K., Buchner, J., (1993b) Small heat shock proteins are molecular chaperones. The Journal of biological chemistry 268, 1517-1520.

re

21. Jena, R., Garg, D.K., Choudhury, L., Saini, A., Kundu, B., (2018) Heterologous expression of an engineered protein domain acts as chaperone and enhances thermotolerance of Escherichia coli. Int J Biol Macromol 107, 2086-2093.

lP

22. Katakam, M., Banga, A.K., (1997) Use of Poloxamer Polymers to Stabilize Recombinant Human Growth Hormone Against Various Processing Stresses. Pharmaceutical Development and Technology 2, 143-149.

na

23. Kaushik, J.K., Bhat, R., (2003) Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose. Journal of Biological Chemistry 278, 26458-26465. 24. King, J., (1997) Refolding with a piece of the ring. Research News.

ur

25. Kolate, A., Baradia, D., Patil, S., Vhora, I., Kore, G., Misra, A., (2014) PEG — A versatile conjugating ligand for drugs and drug delivery systems. Journal of Controlled Release 192, 67-81.

Jo

26. Laksanalamai, P., Maeder, D.L., Robb, F.T., (2001) Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus. Journal of bacteriology 183, 5198-5202. 27. Laksanalamai, P., Pavlov, A.R., Slesarev, A.I., Robb, F.T., (2006) Stabilization of Taq DNA polymerase at high temperature by protein folding pathways from a hyperthermophilic archaeon, Pyrococcus furiosus. Biotechnology and bioengineering 93, 1-5. 28. Lee, S., Kim, J.S., Yun, C.H., Chae, H.Z., Kim, K., (2009) Aspartyl aminopeptidase of Schizosaccharomyces pombe has a molecular chaperone function. BMB reports 42, 812-816.

28

29. Li, K., Jiang, T., Yu, B., Wang, L., Gao, C., Ma, C., Xu, P., Ma, Y., (2013) Escherichia coli transcription termination factor NusA: heat-induced oligomerization and chaperone activity. Sci Rep 3, 2347. 30. Librizzi, F., Carrotta, R., Spigolon, D., Bulone, D., San Biagio, P.L., (2014) α-Casein inhibits insulin amyloid formation by preventing the onset of secondary nucleation processes. The journal of physical chemistry letters 5, 3043-3048. 31. Liebner, R., Mathaes, R., Meyer, M., Hey, T., Winter, G., Besheer, A., (2014) Protein HESylation for half-life extension: Synthesis, characterization and pharmacokinetics of HESylated anakinra. European Journal of Pharmaceutics and Biopharmaceutics 87, 378-385. 32. Makarewicz, T., Kazmierkiewicz, R., (2013) Molecular dynamics simulation by GROMACS using GUI plugin for PyMOL. Journal of chemical information and modeling 53, 1229-1234.

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33. Makarewicz, T., Kaźmierkiewicz, R., (2016) Improvements in GROMACS plugin for PyMOL including implicit solvent simulations and displaying results of PCA analysis. Journal of Molecular Modeling 22, 109.

-p

34. Mohan Murali Achary, V., Patnaik, A.R., Panda, B.B., (2012) Oxidative biomarkers in leaf tissue of barley seedlings in response to aluminum stress. Ecotoxicology and environmental safety 75, 16-26. 35. Nakamoto, H., Vigh, L., (2007) The small heat shock proteins and their clients. Cellular and molecular life sciences: CMLS 64, 294-306.

re

36. Qiu, L.-M., Ma, J., Wang, J., Zhang, F.-C., Wang, Y., (2010) Thermal stability properties of an antifreeze protein from the desert beetle Microdera punctipennis. Cryobiology 60, 192197.

lP

37. Rydeen, A.E., Brustad, E.M., and Pielak, G.J. (2018). Osmolytes and Protein–Protein Interactions. Journal of the American Chemical Society 140, 7441-7444.

na

38. Romero-Romero, M.L., Risso, V.A., Martinez-Rodriguez, S., Ibarra-Molero, B., and Sanchez-Ruiz, J.M. (2016). Engineering ancestral protein hyperstability. Biochemical Journal 473, 3611-3620.

ur

39. Sabbaghian, M., Ebrahim-Habibi, A., Hosseinkhani, S., Ghasemi, A., Nemat-Gorgani, M., (2011) Prevention of thermal aggregation of an allosteric protein by small molecules: Some mechanistic insights. International Journal of Biological Macromolecules 49, 806-813.

Jo

40. Sasahara, K., McPhie, P., Minton, A.P., (2003) Effect of Dextran on Protein Stability and Conformation Attributed to Macromolecular Crowding. Journal of Molecular Biology 326, 1227-1237. 41. Sharp, K.H., Moody, P.C., Brown, K.A., Raven, E.L., (2004) Crystal structure of the ascorbate peroxidase-salicylhydroxamic acid complex. Biochemistry 43, 8644-8651. 42. Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K., (2002) Regulation and function of ascorbate peroxidase isoenzymes. Journal of experimental botany 53, 1305-1319. 43. Szebeni, A., Olson, M.O., (1999) Nucleolar protein B23 has molecular chaperone activities. Protein Science: A Publication of the Protein Society 8, 905-912. 44. The PyMOL Molecular Graphics System, V.S., LLC. 29

45. Tomar, R., Garg, D.K., Mishra, R., Thakur, A.K., Kundu, B., (2013) N-terminal domain of Pyrococcus furiosus l-asparaginase functions as a non-specific, stable, molecular chaperone. Febs j 280, 2688-2699. 46. Zhang, Y., Ge, J., Liu, Z., (2015) Enhanced Activity of Immobilized or Chemically Modified Enzymes. ACS Catalysis 5, 4503-4513.

Jo

ur

na

lP

re

-p

ro of

47. Zhao, Z., Fu, J., Dhakal, S., Johnson-Buck, A., Liu, M., Zhang, T., Woodbury, N.W., Liu, Y., Walter, N.G., Yan, H., (2016) Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nature communications 7, 10619.

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Figure legends

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Fig. 1. Purification and assay of proteins. (A) SDS-PAGE (15%) showing molecular weight marker (lane M), Ni-NTA column purified NPfA as analyzed through coomassie staining (lane 1) and western blot using anti-His antibody (lane 2). (B) SDS-PAGE (15%) analysis of APX (lane 3) and Native-PAGE (10%) showing in-gel activity assay for APX purified protein (lane 4) using 1 mM Nitro Blue Tetrazolium (NBT) as substrate. (C) Fluorescence spectra of WT and Trp mutated NPfA (W60F). (D) Far UV CD profile of WT 31

and Trp muted NPfA. (E) Equilibrium unfolding profile of WT and W60F monitored through far UV CD. (F) Effect of WT and W60F NPfA on APX activity. Activity of APX heated at 40 ℃ in presence of NPfA (WT or W60F) showed similar profiles and was higher than that of control. Results D-F demonstrate that Trp mutation at position 60 did not have any effect on

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structural stability and chaperone activity of NPfA.

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Fig. 2. Effect of NPfA on APX activity. (A) Percentage activity of APX plotted as a function of increasing molar concentration of NPfA and BSA. Activity of APX without NPfA

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was considered as 100%. (B) APX activity plotted as a function of temperature with or without 1:2 molar concentrations of NPfA. The concentration of APX used was 0.4 μg ml-1

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(15 nM) and error bars represent standard error of the mean of 3 independent experiments.

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Fig. 3. Comparison of NPfA with commonly used osmolytes in thermoprotection of APX.

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APX subjected to 40°C and cooled in the presence of NPfA and osmolytes was measured for its (A) Fluorescence intensity at 340 nm and (B) Activity. (C) A comparative profile of

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structure and function of APX in the presence of different additives.

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Fig. 4. Effect of NPfA on IgG antibody activity. ARI-HAb (A) and AMI-HAb (B) activity plotted as a function of increasing temperature with or without 1:26 molar concentrations of

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I-HAb:NPfA. I-HAb activity at 30 °C without NPfA was considered as 100%. Highest

difference was observed at 60 °C where both the I-HAbs lost maximum of their original

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***P < 0.001, ****P < 0.0001).

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activity. The statistical significance was examined using paired t-test (*P < 0.05, **P < 0.01,

34

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Fig. 5. Effect of NPfA on activity and thermal stability of KOD DNA polymerase.

lP

Enzyme activity as a measure of amplicon band intensity on 1% agarose gel after PCR carried out using (A) decreasing concentrations of KOD polymerase with NPfA (+) or without NPfA (-), and with BSA (*) as shown in Figure. (B) With increasing time of

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incubation of KOD Polymerase at 97 °C without NPfA, with NPfA and with BSA as shown in Figure. Corresponding band intensities as determined by densitometric analysis are shown

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alongside. Error bars represent standard error of the mean of 3 independent experiments.

35

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lP

Fig. 6. Effect of NPfA on heat-induced aggregation of APX. The molar ratio of APX and NPfA was 1:2. The buffer system was phosphate buffer saline (PBS) and molar concentration

na

of APX and NPfA were 21.4 µM and 45 µM, respectively. (A) NPfA effectively protects thermal denaturation of APX that was heated at different temperature for 30 minutes,

ur

followed by cooling to 25 ℃. The excitation wavelength was 295 nm and intensity at 340 nm

Jo

was observed as a measure of folded soluble protein. (B) APX (with or without NPfA) was subjected to thermal denaturation with gradual increase in temperature, and the loss in structure was continuously monitored at 340 nm. (C) Protein solutions were incubated at 40 ℃ and aggregation kinetics was monitored through light scattering. The excitation and emission wavelength were 400 nm. (D) Kinetics of the loss of APX structure in presence or absence of NPfA was monitored through fluorescence emission at 40 ℃.

36

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Fig. 7. Interaction studies of NPfA with APX. ITCanalysis of NPfA-APX interaction at 25 ℃ (A) and 40 ℃ (B). In the upper panel the baseline corrected raw data is shown; in the bottom panel the binding isotherm from the integrated thermogram fit using the two-site

37

model in the Origin software is shown. (C) The size-exclusion chromatogram of NPfA-APX

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re

-p

ro of

complex shown with respect to NPfA and APX alone.

Fig. 8. The size distribution of NPfA and APX as measured through DLS. The DLS measurement of each set of protein was carried out at 25 ℃, followed by 10 minutes heating

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at 40 ℃ and eventually cooling it down to 25 ℃ for 10 minutes. The temperature of

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incubation is indicated in the respective figures.

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Fig. 9. Models showing interaction between NPfA and APX and substrate binding to APX active site. (A) Two monomers of NPfA (4ra6) are docked with single monomeric

-p

APX (2xif). (B) The active site residues (Cys 32 and Arg 172) are shown in presence of

na

lP

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ascorbic acid bound to the APX active site.

Fig. 10. Time dependent evolution of variables through MD simulations. Variations in

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(A) Backbone root mean square deviations (RMSD). (B) Gyration radius (Rg) (C) Solvent accessible surface of the active site pocket (SAS) for APX alone at 300 K (Black), 314

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K (Red) and in complex with NPfA at 310 K (Blue) and 314 K (Magenta). (D) Probability distribution of NPfA-APX H-bond network averaged over the entire trajectory for NPfA:APX complex at 300 K (Blue) and 314 K (Magenta).

39

Table 1. Thermodynamic parameters for the interaction between NPfA and APX obtained through ITC. Best fit was obtained in sequential site binding model and Ka (M-1), ΔH (kcal M-1), ΔS (cal mol-1K-1) were determined for each binding site. 40 ℃

25 ℃

N K1 ΔH1 ΔS1 K2 ΔH2 ΔS2

2.1 2.76E6 ± 6.9E6 1.378E4 ± 2.42E 73.5 1.00E5 -2.735E5 ± 5.23E5 -850

1.9 1.32E5 ± 3.1E5 -2956 ± 1.39E3 13.5 1.09E5 ± 2.4E5 7812 ± 2.00E3 49.2

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-p

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Parameters

40