Spectral and thermal properties of novel eye lens ζ-crystallin

Accepted Manuscript Title: Spectral and thermal properties of novel eye lens ␨-crystallin Author: Ajamaluddin Malik Shurog Albogami Abdulrahman M. Al-Senaidy Abeer M. Aldbass Mohammad A. Alsenaidy Shams Tabrez Khan PII: DOI: Reference:

S0141-8130(16)33030-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.04.101 BIOMAC 7465

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

26-12-2016 13-4-2017 16-4-2017

Please cite this article as: A. Malik, S. Albogami, A.M. Al-Senaidy, A.M. Aldbass, M.A. Alsenaidy, S.T. Khan, Spectral and thermal properties of novel eye lens rmzeta-crystallin, International Journal of Biological Macromolecules (2017), http://dx.doi.org/10.1016/j.ijbiomac.2017.04.101 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Spectral and thermal properties of novel eye lens ζ-crystallin

Ajamaluddin Malik1*, Shurog Albogami1, Abdulrahman M. Al-Senaidy1, Abeer M.

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Aldbass1, Mohammad A. Alsenaidy2, Shams Tabrez Khan3

Department of Biochemistry, Protein Research Chair, College of Science, King Saud

Vaccines and Biologics Research Unit, Department of Pharmaceutics, College of

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University, PO Box 2455, Riyadh 11451, Saudi Arabia.

Department, College of Science, King Saud University, Riyadh, Saudi Arabia.

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*Corresponding author

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3Zoology

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Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia

Dr. Ajamaluddin Malik, Department of Biochemistry, Protein Research Chair, College of

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Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. E-mail: [email protected] Tel.: +966-114696241 Fax: +966-114675797

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Abstract Eye lenses are exposed to thermal, solar radiations, dryness that enhances cataractogenesis. Some animal lenses contain novel proteins in bulk quantities. ζ-

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crystallin occurred in three ecologically divergent species, but it’s physiological role not known. The truncated variant of ζ-crystallin causes hereditary cataract. Guinea pig ζcrystallin is temperature-sensitive and rapidly aggregates at 41oC.

Camels adopted to

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survive above 50oC, which raises an interesting question about how it retains lens

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proteins in the soluble state? Here, we have optimized expression and purification of recombinant camel ζ-crystallin. We have studied thermodynamic and spectroscopic properties using orthogonal techniques.

Dynamic multimode spectroscopy results

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showed that camel ζ-crystallin unfolds via single transition with Tm value of 60.8±0.1°C and van't Hoff enthalpy of 714.7±7.1kJ/mol. Thermal-shift assay calculates Tm value of

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62oC at pH 7. Additionally, the conformational stability of ζ-crystallin increases with ionic-strength. The influence of pH on ζ-crystallin was evaluated where the protein was found to be stable in the pH range of 6-9, but its stability drastically decreases below pH

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6. Our results also showed that quaternary structure of ζ-crystallin drastically changed as

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a result of lowering pH. This study provides significant understandings onto the conformational, thermodynamic and unfolding pathway of camel ζ-crystallin.

Keywords: ζ-crystallin; Cataract; Thermal shift assay; Dynamic multimode spectroscopy

Abbreviations: Amp, ampicillin; CV, column volume; DMS, Dynamic multimode spectroscopy; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatography; Imax, intensity maxima; IPTG, Isopropyl β-D-1thiogalactopyranoside; L, liter; LB, Luria-Bertani; Ni-NTA, Nickel-nitrilotriacetic acid; OD600, optical density at 600 nm; rpm, rotation per minute; Tm, melting temperature;

λmax, wavelength maxima.

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1.

Introduction

The eye lens is specialized transparent tissue, designed to provide clarity to the vision throughout life. The main function of the eye lens is to transmit light and focus it on the retina [1, 2]. The composition and properties of the eye lens is unique. The eye lens

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proteins are synthesized embryonically and cellular organelles are degraded in a

synchronic way to avoid light scattering in the lens [3, 4]. Lens proteins represent 30-

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35% of the total mass; the remaining 65-70% is water compared to 95 % water found in

non-lenticular cells [5]. The main constituents (~90%) of the lens proteins are water

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soluble structural proteins, referred to as crystallins [2, 6]. The transparency and high refractive index of mammalian lenses are due to very high concentrations of crystallins in the lens fiber cells [5]. Crystallins according to the species of origin could be divided into

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two types. First, crystallins in vertebrates, with ubiquitous main components consisting of α-, β- and γ- superfamilies. Second, crystallins in the lenses of specific species, named

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taxon-specific crystallins, which include δ, ε, λ, ζ, ρ, τ [7, 8].

Taxon-specific crystallins are metabolic enzymes with catalytic functions in non-

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lenticular tissues [9]. Several metabolic enzymes in different species are expressed in

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large quantities in the eye lens and this concept has been referred to as gene sharing [10]. One of such taxon-specific crystallins, ζ-crystallin (NADPH-dependent quinone oxidoreductase), abundant in large quantities in the lenses of three ecologically divergent animals: camelids, certain hystricomorph rodents and Japanese tree frog [11-13]. ζcrystallin constitutes nearly 10 % of the water-soluble crystallin in the guinea pig and camel eye lens [14, 15]. Interestingly, ζ-crystallin is also present in several other eye lenses, including humans, however, in lower catalytic amounts [16].

ζ-crystallin catalyzes the reduction of quinones via one-electron transfer in the presence of NADPH [17]. It is a homo-tetrameric protein with a molecular weight of 35 kDa. The physiological importance of the oxidoreductase activity of ζ-crystallin in the lens is not clear. However, its role in cataractogenesis has been viewed in previous studies. In one study, it has been found that a truncated mutant (N-terminal 34 amino acid deletion) of ζ-

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crystallin caused hereditary nuclear cataract in the genetic strain line 13 / N guinea pig [18]. Like different quinone reductases, ζ-crystallin may function as a detoxification tool via two mechanisms: First, since the enzyme exhibits a high affinity toward NADPH, the enzyme may bind to NADPH to preserve the reducing environment in the lens and

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protects it from oxidative damage [19]. Second, the oxidoreductase activity may also function as a defense against quinones and different oxidizing agents [17].

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Camel’s ζ-crystallin is 87 and 83% identical to humans and guinea pigs ζ-crystallins,

respectively. The natural habitat of guinea pig is mild and it’s ζ-crystallin is thermally

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sensitive that rapidly aggregates at 41 oC [20]. On the other hand, dromedary camels (Camelus dromedarius) were domesticated some 5,000 years ago in the Arabian

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Peninsula. Dromedary camels live under the extremely harsh climate conditions of hightemperature, UV-radiation, dryness and scarcity of food [21]. Strong positive associations

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have been established between environmental factors such as heat, dehydration, and UV radiation and the process of cataractogenesis [22-26]. The eye lens proteins are thermally-sensitive [26]. An overnight incubation of intact human, pig and rabbit eye

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lenses at 50 °C causes severe damage equivalent to four decades of aging [26-29].

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Unfolding and aggregation of the eye lens proteins increases light scattering and results in lens opacification, known also as cataract [30].

In the extreme desert climates (>50 °C and severe dryness), the mortality rates of adult camels are lower compared to other animals [31]. Since the Arabian camel is so well adapted to live in extreme desert climatic condition of heat, solar radiation and acute physiological stress of water, it is interesting to understand its lens protective mechanisms. In this study, we overexpressed ζ-crystallin in E.coli and purified it to homogeneity using two-step chromatography. We investigated the thermodynamics and spectroscopic properties using dynamic multimode spectroscopy technique. Our result shows that ζ-crystallin unfolds via a single transition with Tm value of 60.8 ± 0.1 °C. We have also used an orthogonal technique based on intrinsic tryptophan fluorescence to study the thermal stability of ζ-crystallin at different ionic strengths and pH conditions.

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Our results showed that ζ-crystallin undergoes a single thermal transition at pH conditions between 4 and 9 and unfolds via a pathway of two-state folding. At pH 7.0, ζcrystallin was stabilized in the presence of 0-500 mM NaCl. The Tm value of ζ-crystallin varies from 62.4-64.5 oC between pH 6-9. But, the thermal stability decreased drastically

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below pH 6. At pH 5, tetrameric ζ-crystallin first converted into a quaternary structure with lighter molecular weight, whereas at lower pH conditions (3.5) it converted into a soluble oligomer. Further reduction in pH leads to more aggressive aggregation. This

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study will not only help in the understanding of the thermal and spectroscopic properties

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oxidoreductases with the similar structural scaffold.

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of camel ζ-crystallin alone but also could help shed some light onto other quinone

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2.

MATERIALS AND METHODS

2.1

Materials

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Plasmid pET15b ζ-crystallin encoding ζ-crystallin has been reported elsewhere [32].

Expression host strain E. coli BL21 (DE3) pLysS was obtained from Life Technologies.

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IPTG, benzonase, ampicillin, PMSF and NADPH were obtained from Sigma. Chicken

egg lysozyme was purchased from Fluka. 9,10-phenanthrenequinone obtained from

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Merck. HisTrap HP, Superdex 200 prep grade, AKTA purification system were purchased from Amersham Biosciences. All other chemicals used in this study were reagent grade. 4-20 % precasted SDS–PAGE gradient gel was from Genscript. Circular spectroscopy

(CD)

from

Applied

Photophysics.

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dichroism

Cary

eclipse

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spectrofluorometer was from Agilent Technologies.

Optimization of camel lens ζ-crystallin expression

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In this study, ζ-crystallin was expressed in E. coli BL21 (DE3) pLysS strain. Throughout

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study 200 µg/mL ampicillin was used in a liquid (LB) and solid media (LBagar) and incubated over-night in an incubator shaker at 37 °C at a speed of 120-150 rpm. In this study, different parameters were optimized to increase the specific and volumetric yield of ζ-crystallin. To optimize post-induction growth temperature, 20 ml pre-cultures were induced with 1 mM IPTG at the mid-log phase and cultures were grown at 24, 30 and 37 o

C for 3 hours. The expression level of recombinant ζ-crystallin in soluble cell extract and

pellet fractions were analyzed on 4-20 % gradient SDS-PAGE. In the next experiment, when pre-cultures (20 ml) growth was reached in the mid-log phase, different concentrations of IPTG (0, 10, 100, 250, 500 and 1000 mM) were added and harvested after 3 hours of growth at optimum temperature (30 oC). Again, the yield of soluble ζcrystallin was analyzed on SDS-PAGE. Subsequently, the post-induction incubation time was optimized by inducing culture at mid-log phase with optimum IPTG concentration (10 µM) and grown at optimum temperature (30 oC) for 0, 1, 2, 3, 5 and 24 hours. The yield of soluble ζ-crystallin was analyzed by SDS-PAGE. Under optimum condition (30

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C growth temperature, 10 µM IPTG and 5 hours of post-induction incubation time), liter

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scale expression experiments were set up to prepare biomass.

Soluble protein extraction and purification of ζ-crystallin

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Soluble protein from biomass was extracted using gentle chemical lysis method as

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described in [32]. The Clear lysate was passed through His-trap HP 1 mL column using Ä KTA purification system. The column was first equilibrated with 10 CV buffer A (50

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mM Tris pH 8.0, 500 mM NaCl, 20 % glycerol, and 10 mM imidazole) at 1 ml/min flow rate. Cell lysates were then passed through column at 1 ml/min flow rate using peristaltic pump. Column was washed with 5 CV buffer A to remove unbound proteins. To elute

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bound protein, gradient was set up to 500 mM of imidazole buffer B (buffer A containing 500 mM imidazole). Fractions were collected and crude extract, flow through, wash were

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analyzed on SDS-PAGE. The enriched fractions of ζ-crystallin eluted from Ni-NTA column were pooled and further purified by Superdex 200 gel filtration column. The column was first equilibrated with 2 CV of 10 mM Tris pH 8.0, 100 mM NaCl, and 20 %

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glycerol, and then Ni-NTA elute was loaded on the column using a superloop. Fractions

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were collected and purity was analyzed on SDS-PAGE. Highly pure fractions were combined and stored at -80 oC.

2.4

Protein Quantification

Glycerol was removed by extensive dialysis against 20 mM phosphate buffer pH 7.0 containing 100 mM NaCl before each biophysical study. The concentration of pure ζcrystallin was estimated spectrophotometrically at 280 nm using molar extinction coefficient of 21,890 M-1 cm-1.

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2.5

Enzymatic activity assay

To examine the native folding of ζ-crystallin, the NADPH quinone oxidoreductase activity was assayed according to the protocol [15]. The assay was done in 20 mM

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sodium phosphate buffer pH 7.8, 0.2 mM EDTA containing 100 µM NADPH as a cofactor, 25 µM PQ as a substrate, and 1 µg of the ζ-crystallin enzyme in a total volume

of 1 ml. The change in absorbance was measured at 340 nm. A blank test was also done

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2.6

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in identical condition, except the addition of ζ-crystallin.

Study of thermal unfolding by dynamic multimode spectroscopic technique

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ζ-crystallin was diluted to 0.2 mg/ml in 20 mM phosphate buffer, pH 7.0. A thermal probe inserted in the short path length (1 mm) cuvette. DMS was performed using a

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Chirascan-Plus spectrophotometer equipped with Peltier temperature controller as described in [33]. Temperature-dependent changes in the ζ-crystallin conformational were measured at the temperature ramp set from 20 to 94 oC, at a rate of 1 oC/min. The

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far-UV CD spectra were collected between 200 and 250 nm. Global 3 software provided

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by Chirascan’s manufacturer was used for processing thermal transition data.

Spectral and thermal studies by fluorescence spectroscopy

Fluorescence spectra of ζ-crystallin (50 µg/ml) at different pH conditions (pH 4-5 in 20 mM acetate buffer; pH 6-8 in 20 mM phosphate buffer and pH 9 in 20 mM GlycineNaOH buffer) were recorded using Cary Eclipse fluorescence spectrophotometer using a 10 mm path length cuvette. To measure fluorescence, ζ-crystallin was excited at 280 nm and emission spectra were collected between 300-400 nm (excitation 5 nm and emission 10 nm bandwidths) at 25 oC. Temperature melting studies of ζ-crystallin were done at a linear increase of 1 oC/min. The temperature inside protein samples was monitored using an internal temperature probe. To evaluate the effect of pH on the thermal stability of ζcrystallin, samples at different pH conditions were gradually heated from 20 to 90 oC. Tryptophan fluorescence was measured by exciting at 295 nm (5 nm bandwidth) and

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collecting at emission intensities of 330 and 350 nm. The ratio of fluorescence intensity at 350 nm to 330 nm was obtained. The melting curves were fitted using Sigma plot software and plotted as a function of temperature. The effect of ionic strength on ζcrystallin thermal stability was studied by incubating 50 mM ζ-crystallin with 0, 50, 100,

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150, 250 and 500 mM NaCl in 20 mM phosphate buffer at pH 7.0. Thermal shift assays were performed as described above.

Effect of pH on the quaternary structure.

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2.8

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Superdex 200 Increase 10/300 GL, a gel permeation column, with fractionation range 10600 kDa was calibrated with five proteins of different molecular weights (Thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa and ovalbumin 43

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kDa). ζ-crystallin (1 mg/ml) was incubated at different pH conditions containing 100 mM NaCl (50 mM Glycine-HCl buffer pH 9.0, 50 mM Phosphate buffer pH 7.0, 50 mM

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Acetate buffer pH 3.5 and 5.0) for two hours at 4 oC. The column was equilibrated with respective buffers and equal amounts of ζ-crystallin were loaded on the column. The

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protein was eluted at 1ml/min flow-rate at room temperature.

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RESULT AND DISCUSSION

3.1

Optimization of recombinant ζ-crystallin expression

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3.

Codon optimized Camelus dromedaries CRYZ gene was cloned into a pET15b vector,

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under the control of T7 promoter [32]. To facilitate purification, hexahistidine tag fused

at the N-terminus of ζ-crystallin was used. A specific cleavage thrombin site was placed

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between His-tag and ζ-crystallin to remove his-tag after purification if required. The fusion protein was 349 amino acids in length, corresponding to 37.3 kDa. Camel lens ζcrystallin expression in E.coli was aggregation-prone when expressed under un-optimized

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conditions [32]. Therefore, we optimized critical expression parameters (growth temperature, inducer concentration and post-induction incubation period) to increase the

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specific and volumetric yield of soluble ζ-crystallin.

Camel lens ζ-crystallin was expressed in E. coli BL21(DE3) pLysS strain as described in

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detail in the “Materials and Methods” section. E. coli BL21(DE3) pLysS is a lysogenic

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strain. The λ-DE3 cassette was inserted in the chromosome which expresses highly active T7 RNA polymerase under the control of the IPTG-inducible lac-operon promoter. Also, this strain harbor pLysS plasmid which suppresses the leakiness of ζ-crystallin under uninduced condition. [34]. Typically, the optimum temperature for recombinant protein production in E. coli is 37 °C and some other studies report a 37 °C as the best temperature for maximum protein production [35, 36]. Several studies showed that rate of expression and culture temperature would have an effect on the correct folding of recombinant proteins and inclusion body formation [37, 38]. This means that the production of soluble protein can be increased by simply lowering the growth temperature during protein synthesis, which lowers aggregation of the target protein [38, 39]. For recombinant ζ-crystallin, when an expression temperature of 37 °C was used, more ζ-crystallin presented in the insoluble pellet fraction. However, lowering the expression temperature to 30 °C resulted in an increase in the yields of ζ-crystallin in its soluble form. This could be explained as reduced temperatures would lead to slower rates

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of transcription and translation and would minimize hydrophobic interactions that cause the formation of inclusion bodies. We found that when culture was induced with 10 µM IPTG and incubated at 30 oC for 5 hours, the yield of soluble ζ-crystallin was highest (data not shown). Purification of ζ-crystallin was achieved using two-step Soluble

ζ-crystallin

his-tagged

was

captured

using

Ni-NTA

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chromatography.

chromatography (Fig. 1). Fractions containing soluble ζ-crystallin were pooled and

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polished using gel filtration chromatography (Fig. 2). We obtained nearly 23 mg of highly soluble ζ-crystallin from 1L culture, nearly 5-fold increase in yield from a

Thermodynamic Characterization of recombinant ζ-crystallin

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3.2

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previous study [32].

Circular dichroism is an excellent technique for rapid determination of the secondary and

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tertiary structure and folding properties of proteins. Dynamic multimode spectroscopy based on far-UV CD is an information-rich experimental technique, which provides insight into conformational changes as a function of temperature. To calculate the

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thermodynamics of unfolding and the mid-points of unfolding transitions, far-UV CD

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spectra between 200 and 250 nm of ζ-crystallin was recorded using temperature ramp of 1 oC/min. Fig. 3A shows that ζ-crystallin undergoes a single, sharp, well-defined thermal transition as the temperature is increased, suggesting two folding states (a native and denatured state without folding intermediates). Maximum shift in CD intensity was observed at 218 nm and a reduction in CD signal intensity was observed for ζ-crystallin as its secondary structure unfolds as we raise the temperature. However, it retains some residual secondary structure in its denatured form at the highest temperature tested (Fig. 3 B). The relative concentration of the folded ζ-crystallin species at the temperature range tested is shown in Fig. 3 C. The thermal melting point (Tm) and van't Hoff enthalpy of camel lens ζ-crystallin were 60.8 ± 0.1 °C and 714.7 ± 7.1 kJ/mol, respectively. A 3D model of the thermal transition in ζ-crystallin was obtained using Global 3 analysis software (Fig. 3 D). A single transition as a function of temperature is clearly apparent at lower wavelengths. Overall, our results indicate that in term of secondary structure, camel ζ-crystallin is more stable compared to guinea pig’s ζ-crystallin. Despite the high

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homology (83 %) between both sequences, ζ-crystallin of guinea pig,aggregates at nearly 20 oC lower compared to camel ζ-crystallin [20]. ζ-crystallin is a member of a large alcohol dehydrogenase family found in many organisms. Compared to camel ζ-crystallin, one study showed that yeast ADH unfolds via a single transition with a Tm value of 65.5

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◦C [40], while in another study using S. cerevisiae’s quinone oxidoreductase Lot6p

reported a Tm of 58.9±0.4 ◦C [41] reflecting variable stability profiles among different

3.3

Effect of pH on ζ-crystallin fluorescence

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species.

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The intrinsic fluorescence spectrum of ζ-crystallin exhibit a blue shifted λmax at 315 nm (Fig. 4). Highest fluorescence Imax was observed at pH 7, however, at more basic pH

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conditions, fluorescence Imax decreased significantly, while λmax remained unchanged at the pH conditions tested. In earlier studies, λmax of camel’s and guinea pig’s ζ-

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crystallin was reported to be at 315 and 312 nm, respectively [19, 42]. The unusual blueshift in fluorescence emission of ζ-crystallins can be attributed to the tyrosine richness

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and localization of a single tryptophan in a highly non-polar environment. Camel (EMBL accession no: GADU01072870) and guinea pig lens ζ-crystallins (NP_001166407.1) accommodate 11 and 10 tyrosine residues, respectively. In one study, camel’s ζ-crystallin under denaturing conditions using increasing concentrations of urea, red shifts by 40 nm [19]. Generally, for proteins containing tyrosine and tryptophan residues, fluorescence spectra is dominated by tryptophan fluorescence. However, tryptophan spectral dominance in some cases is phenomenal as in the case of human serum albumin which accommodates just one tryptophan and seventeen tyrosines. [43]. Blue-shifted tryptophan fluorescence has been observed in other proteins as well. P. fluorescens Apoazurin which accommodate a single tryptophan that is completely buried in the hydrophobic core of the protein, exhibited a λmax of 308 nm which is the smallest Stokes shift known for tryptophan accommodating proteins [44]. In another example concerning ribonuclease

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T1, a λmax of 325 nm was reported where the tryptophan was partially buried in a non-

Effect of pH on thermal stability of ζ-crystallin

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3.4

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polar environment [45].

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Intrinsic fluorescence spectroscopy is a highly sensitive tool, as it provides information about the microenvironment of Trp and Tyr residues within proteins. Tyrosine emission maximum is less sensitive to its local environment compared to tryptophan [45, 46]. The

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induced sensitivity behavior of tryptophan stems from the fact that its indole fluorophore undergoes two nearby isoenergetic transitions, unlike tyrosine which undergoes through a

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single electronic state [46]. The polarity of the fluorophores' microenvironment changes during protein unfolding which in turn leads to changes in the fluorescence intensity (Imax) as well as fluorescence maxima wavelength (λmax). Thus, intrinsic tryptophan

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fluorescence is a good readout of tertiary structure and could detect subtle protein

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conformational changes in solution. It has frequently been used for the characterization of protein’s conformational changes under different stress conditions [47, 48]. To obtain the temperature-melting curves, camel lens ζ-crystallin was gradually heated from 20-90 oC at a rate of 1 oC/min. The ratio of 350/330 nm tryptophan fluorescence was plotted as a function of temperature at the different pH conditions tested (Fig. 5a). The fluorescence data was fitted following the equation f= y0+a/(1+exp(-(x-x0)/b)) with r2 value of 0.9910. The ratio of 350/330 nm is preferred for most proteins since the ratio monitors the change in tryptophan emission intensity as well as the shift of λmax towards higher (“redshift”) or lower wavelengths (“blueshift”). Camel lens ζ-crystallin was moderately stable, with Tm value of 62 oC. During thermal unfolding it undergoes a single transition and, as in the case of secondary structure, its tertiary structure unfolds via twostate unfolding pathway at all pH conditions. ζ -crystallin retains stability at pH conditions ranging from 6-9. However, a reduced conformational stability at pH 4 was

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detected as the Tm drastically is reduced to 42.1 oC (Fig. 5b). ζ-crystallin aggregates below pH 4, therefore, we could not evaluate the thermal stability at these conditions.

The effect of ionic strength on the ζ-crystallin stability was measured by thermal-shift

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assay using intrinsic tryptophan fluorescence. Temperature melting curves were obtained by gradually heating 50 µg/ml of ζ-crystallin in the presence of increasing NaCl salt concentrations (0-500 mM) from 30-90 oC at a rate of 1 oC/min. Our result showed that

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ζ-crystallin was slightly stabilized (~3 oC) in the presence of 500 mM NaCl (Fig. 6). The

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ionic strength of the solution affects the protein stability by Coulomb-driven residue pairing [49, 50]. Depending upon the distribution of the specific charges around the protein, it may be slightly stabilized or destabilized by salts. It has been observed earlier

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that mesophilic proteins are stabilized in the presence of salts while thermophile and hyperthermophile proteins are destabilized under higher salt concentration [51]. Also, it

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was found that halophilic proteins are destabilized under low salt concentrations [52, 53]. These proteins are adapted to survive under high salt concentrations by displaying relatively large number of negatively charged residues on the surface [53, 54]. These

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extremophiles proteins are adapted to maintain the right conformation and function

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through electrostatic interactions [51].

ζ-crystallin is an acid-labile protein which loses conformation and aggregate at acidic conditions (Fig. 5). Therefore, we investigated the effect of varying pH from 3.5 to 9.0 on its quaternary structure using gel permeation chromatography. At pH 7.0, ζ-crystallin eluted as a single peak, corresponding to its tetrameric state [32], as seen in the pink chromatogram in Fig. 7. When ζ-crystallin was incubated at pH 9 (cyan colour), it went through a slight conformational change and a small population of it was observed at a dimeric state. Incubating ζ-crystallin at an acidic condition of pH of 5.0, however, lead to a huge shift in the quaternary structure equilibrium as seen in the blue chromatogram. The protein eluted in a shifted peak with a shoulder corresponding to an equilibrium shift to a trimeric and dimeric states. Further lowering the pH leads to the formation of a soluble oligomer (red chromatogram). ζ-crystallin at pH 3.5 eluted in the void volume as

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a sharp, single peak, indicating conversion of its tetrameric form to a soluble oligomeric.

Conclusions

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4.

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As shown in fig 5b, ζ-crystallin stability rapidly decreased with increasing acidity.

We have optimized conditions to increase the yield of soluble ζ-crystallin in E.coli and

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purified it to homogeneity. Two orthogonal techniques based on far-UV CD (secondary structure of proteins) and intrinsic fluorescence (tertiary structure) were used for studying

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the unfolding pathway of ζ-crystallin and measured its spectroscopic and thermodynamic properties. The results indicated that ζ-crystallin undergoes a single thermal transition and follows a two-state pathway of unfolding at all pH conditions tested. The DMS and

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thermal shift assay methods calculated a Tm value of 60.8 ± 0.1 and 62 oC at pH 7.0, respectively, in agreement with sequential unfolding events of secondary and tertiary

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structures. Camel lens ζ-crystallin is an acid-labile protein whose stability decreases

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below pH 6 and starts forming aggregates below pH 4.0. Similar folding behavior has been observed in the alcohol dehydrogenase family to which ζ-crystallin protein belongs. For example, yeast alcohol dehydrogenase and S. cerevisiae quinone oxidoreductase Lot6p unfold via single transition with Tm values of 65.5◦C and 58.9±0.4 ◦C, respectively, indicating similarity in the folding pathway within the alcohol dehydrogenase proteins scaffold. In light of our results, we plan next to evaluate the effect of different factors such as NADPH and different low molecular weight additives on the conformational stability of camel lens ζ-crystallin.

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Conflict of interest

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To the best of our knowledge, no conflict of interest, financial or others, exists. All authors are fully aware of this submission.

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Acknowledgements

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This study was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz city for Science and Technology, Kingdom of Saudi Arabia, Award Number (12-MED-2932).

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Figure Legends

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Figure 1. Purification of His-tagged ζ-crystallin via Ni-NTA column. (A) Bound ζcrystallin was eluted with linear gradient of imidazole up to 500 mM. The purity of four fractions labeled on the peak was analyzed using SDS-PAGE (B) SDS-PAGE analysis of His-tagged ζ-crystallin purification. Lane 1, low molecular weight marker; lane 2, crude soluble extract; lane 3, flow-through; lane 4, wash; lane 5, fraction 1; lane 6, fraction 2; lane 7, fraction 3 and lane 8, fraction 4.

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Figure 2. Purification of ζ-crystallin via Gel filtration column. (A) Pooled fractions from Ni-NTA column was loaded on Superdex 200 column. A single symmetrical peak was pooled. (B) Purity analysis by SDS-PAGE. Lane 1, low molecular weight marker and lane 2, the pool of fractions.

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Figure 3. Dynamic Multi-mode spectroscopic analysis of ζ-crystallin. (A) Temperature-dependent conformational changes in ζ-crystallin at different wavelengths measured in the far-UV CD region from 200-250 nm. B. Measured far-UV CD spectra of the contributing species. The solid line showed native recombinant ζ-crystallin while the dotted line showed residual structure left in the thermally denatured ζ-crystallin. C. Calculated concentration profiles of the folded species. The solid line showed native recombinant ζ-crystallin while the dotted line showed the thermal unfolded state. D. Calculated temperature, wavelength and CD surface. The 3D model was calculated using Global 3 software through CD values obtained at increasing temperatures in the far-UV region. Samples were prepared in 20 mM sodium phosphate buffer at pH 7.0 containing 20 mM NaCl. Fig 4. Fluorescence spectra of camel lens ζ-crystallin at different pH conditions. ζcrystallin (50 µg/ml) at different pH conditions was excited at 280 nm and data was collected from 300-400 nm at 25 oC. Each peak is labeled with the pH condition tested. Fig. 5. Thermal shift assay using tryptophan fluorescence. (A) ζ-crystallin at different pH conditions was continuously heated from 20-90 oC at a rate of 1 oC/min. Samples

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were excited at 295 nm and emission at 330 and 350 nm were recorded. The ratio of 350 nm/330 nm was curve fitted and plotted as a function of temperature. ζ -crystallin unfolds via a single transition at pH conditions of 4-9 and the mid-point of transition identified as thermal melting point (Tm). (B) Thermal melting point (Tm) of ζ-crystallin at the different pH conditions tested was plotted as a function of pH.

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Fig. 6. The stability of ζ-crystallin at different ionic strength. ζ-crystallin at different concentrations of NaCl (0-500 mM) at pH 7.0 was continuously heated from 30-90 oC at a rate of 1 oC/min. Samples were excited at 295 nm to record tryptophan emission at 330 and 350 nm. The 350/330 nm ratio at different ionic strength (red circle, 0 mM NaCl; green, 50 mM NaCl; blue, 100 mM NaCl; pink, 150 mM NaCl; cyan, 250 mM NaCl and black, 500 mM NaCl) was plotted as a function of temperature. ζ -crystallin follows two state folding. The thermal melting point (Tm) as a function of ionic strength was plotted in the inset.

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Fig. 7. Quaternary structure of ζ-crystallin at different pH. ζ-crystallin at different pH conditions was passed through superdex 200 column (fractionation range 10-600 kDa). ζcrystallin at pH 7.0 (pink color) was eluted as a single peak corresponding to its tetrameric form. At alkaline pH 9.0 (cyan), a small peak appeared indicating the presence of small dimeric population along with the tetrameric form. At slight acidic pH 5.0, tetrameric form was dissociated into trimeric and dimeric forms (blue). When the pH was lowered to 3.5, tetrameric ζ-crystallin converted to a soluble aggregate that eluted in the void volume (red).

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