Structure and Functional Properties of a Multimeric Protein αA-Crystallin Adsorbed on Silver Nanoparticle Surface

Proteins adsorb onto a nanoparticle surface to form a protein–nanoparticle corona which becomes the identity of the nanoparticle in the cellular environment. Conformation of the protein at the interface influences the cellular uptake of the nanoparticle. Hence, interaction of proteins with nanomaterials is of special significance in the field of biotechnology. Adsorption of protein on the nanoparticle surface is a complex process that depends on the dielectric properties and pH of the medium, surface morphology and surface heterogeneity of the nanoparticle, and the quaternary structure of the protein. Thus, interaction of a large multimeric protein with a nanoparticle will be different from that of small oligomeric proteins. In this article we report the conformational and functional properties of a large oligomeric protein αA-Crystallin, a major constituent of the mammalian eye lens, adsorbed onto silver nanoparticle surface. Selective alkylation of the two cysteine residues at the α-Crystallin domain, followed by ITC study showed that these residues play crucial roles in the interaction process. The chaperone function and the refolding capacity of the protein, which is primarily governed by the α-Crystallin domain, are lost to a significant extent when adsorbed onto AgNP surface. The protein in the interface also shows loss of oligomerization that is linked to the biological activity of the protein. Nonetheless, the protein at bio–nano interface shows resistance to urea unfolding process as compared to protein in the solution phase. This might be due to the coordination of AgNP with two cysteine residues of β8 and β9 region of the α-Crystallin domain that imparts extra stability. The compactness in the structure of the adsorbed protein reduces the dynamics of the subunit exchange, which was confirmed by the FRET study. The secondary structure of αA-Crystallin bound to AgNP at substoichiometric ratio remained native-like

Analysis of changes in the secondary structure compositions (in percentage) of full length and the N terminus truncated forms of recombinant purified Pol λ after UV-B and high salt stress by far-UV CD spectroscopy.

<p># (Errors represent SD from triplicate measurements)</p><p>Analysis of changes in the secondary structure compositions (in percentage) of full length and the N terminus truncated forms of recombinant purified Pol λ after UV-B and high salt stress by far-UV CD spectroscopy.</p

Changes in surface hydrophobicity of purified recombinant full length and N terminus deletion mutants of Pol λ following UV-B irradiation or high salt treatment.

<p>(A) Bis-ANS binding titration of untreated control, UV-B irradiated and high NaCl treated recombinant Pol λ. (B) Bis-ANS binding titration of untreated control, UV-B irradiated and high NaCl treated Del 2 protein. (C) Bis-ANS binding titration of untreated control, UV-B irradiated and high NaCl treated Del 3 protein. (D-F) Representative Scatchard plots of untreated control, UV-B irradiated and high NaCl treated recombinant Pol λ, (G-I) Del 2 protein and (J-L) Del 3 protein for the determination of stoichiometry (<i>n</i>) and the dissociation constant (k_d) respectively. 0.02 mg/mL of each purified protein in 50 mM Tris-HCl buffer, pH 7.5 (containing 1 mM PMSF and 1 mM β-mercaptoethanol) was titrated by addition of aqueous solution of Bis-ANS. The excitation and emission wavelengths were 390 and 490 nm, respectively. The intensities at 490 nm, from the titration, were plotted as a function of Bis-ANS concentration. UV-B and NaCl treatment of purified recombinant proteins were carried out as described under ‘Materials and Methods’.</p

Pol λ interacts with HSP90 <i>in vivo</i>.

<p>(A) Leaves from <i>Agro</i>-infiltrated tobacco (<i>N</i>. <i>benthamiana</i>) plants, transiently and ectopically expressing AtPolλ-C-TAP (DKLAT1G10520.1) and AtHSP90.1-C-TAP (DKLAT5G52640) were harvested and cross-linked with 1% formaldehyde. Protein extracts from the harvested tobacco leaves was immunoprecipitated with affinity purified anti-AtPolλ polyclonal antibody and anti-AtPolλ immunoprecipitate (IP) was probed with anti-HSP90 antibody (1:500 dilution) (coIP). (B) Pol λ and HSP90 interacts in <i>Arabidopsis</i> at endogenous levels. <i>Arabidopsis</i> seedlings were grown for 7–10 under long day cycles, seedlings were harvested, cross-linked with 1% formaldehyde and protein extract was prepared. The protein extract was immunoprecipitated with affinity purified anti-AtPolλ polyclonal antibody and immune complexes from protein extracts were probed for endogenous AtPolλ (IP) and coimmunopreciptated endogenous HSP90 (coIP) using affinity purified anti-AtPolλ polyclonal antibody (1:250 dilution) and anti-HSP90 antibody (1:500 dilution), respectively. Actin, detected by anti-actin monoclonal antibody was used as loading control. Protein extracts from <i>atpolλ-3</i> was used as positive control while <i>atpolλ-1</i> was used as negative control. <i>atpolλ-1</i> is a null mutant line, devoid of Pol λ expression and <i>atpolλ-3</i> is Pol λ protein positive mutant allele. Migration positions of Pol λ or HSP90 have been indicated. (C) Geldanamycin (GDA) treatment inhibits Pol λ-HSP90 interaction. 7-days-old <i>Arabidopsis</i> seedlings (Col-0) were treated with 5 μM GDA or vehicle (1% DMSO) for 8 h, protein extracts prepared and immunoprecipitated with anti-AtPolλ polyclonal antibody. Immune complexes from protein extracts were probed for endogenous AtPolλ (IP) and coimmunopreciptated endogenous HSP90 (coIP). Actin was used as loading control. (D) and (E) Geldanamycin treatment reduces Pol λ protein level. 7-days-old <i>Arabidopsis</i> seedlings (Col-0) were treated with 1% DMSO (D) or 5 μM GDA (E) for the indicated time points and protein extracts were immunoblotted as indicated. Actin was used as loading control. Quantifications of the data in (D) and (E) (lower graph). *<i>P</i> <0.05, *<i>P</i> <0.01 relative to respective controls (<i>n</i> = 3). (F) 7-days-old wild-type <i>Arabidopsis</i> seedlings were treated with 5 μM GDA for 8 h with vehicle (DMSO) or with 50 μM MG132 for at least 4 h. Protein extracts were then immunoblotted using anti-AtPolλ polyclonal antibody. Actin was used as loading control. (G) 7-days-old wild-type <i>Arabidopsis</i> seedlings were treated with 200 μM cycloheximide in the absence (upper panel) or presence of 5 μM GDA for the indicated time points (lower panel) and protein extracts were immunoblotted as indicated. (H) Pol λ signals were quantified and normalized against actin signals. Data shown are means ± SD of three independent replications (lower graph). Representative gel images from at least three independent trials are shown.</p

Understanding the Physical and Molecular Basis of Stability of <i>Arabidopsis</i> DNA Pol λ under UV-B and High NaCl Stress

<div><p>Here, we have investigated the physical and molecular basis of stability of <i>Arabidopsis</i> DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. <i>In vivo</i> aggregation studies using transient expression systems in <i>Arabidopsis</i> and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.</p></div

Acrylamide and KI quenching constants (K_SV) of control, UV-B irradiated and high salt treated purified recombinant full length and N terminus deletion versions of recombinant purified <i>Arabidopsis thaliana</i> DNA Pol λ.

<p># (Errors represent SD from three independent measurements)</p><p>Acrylamide and KI quenching constants (K_SV) of control, UV-B irradiated and high salt treated purified recombinant full length and N terminus deletion versions of recombinant purified <i>Arabidopsis thaliana</i> DNA Pol λ.</p

Thermodynamic stability of recombinant full length and the N terminus truncated forms of Pol λ under UV-B and high salt stress.

<p>(A-C) Urea induced denaturation profiles of recombinant full length Pol λ without treatment (A), after UV-B irradiation (B) or high salt treatment (C), respectively. (D-F) Urea induced denaturation profile of Del 2 protein without treatment (D), after UV-B irradiation (E), and high salt treatment (F). (G-I) Urea induced denaturation profile of DNA Pol λ-Del 3 protein without treatment (G) or after UV-B irradiation (H) and high salt treatment (I). The experiments were performed using 0.05 mg/mL of purified protein in 50 mM Tris-HCl buffer, pH 7.5 (containing 1 mM β-ME and 1 mM PMSF). Untreated control, UV-B irradiated (~200 J/m² UV-B irradiation for 4 h at 25°C) and high salt (500 mM NaCl treatment for 2 h at 25°C) treated protein samples were incubated in presence of increasing concentrations of urea (0–8 mM) in total 600 μL of reaction volume for about 16 h at room temperature (25°C). Samples were excited at 295 nm and emission was scanned between 300 to 400 nm. Ratio of fluorescence intensity at 337 nm to the same at 350 nm was plotted as function of urea concentration.</p

Tryptophan fluorescence spectra of UV-B irradiated purified recombinant full length and N terminus truncated forms of <i>Arabidopsis</i> DNA Pol λ (AtPolλ).

<p>0.05 mg/mL of purified (A) full length recombinant Pol λ, (B) Del 1, (C) Del 2, and (D) Del 3 protein samples in a final volume of 600 μL of 50 mM Tric-HCl buffer, pH 7.5 (containing 1 mM β-ME and 1 mM PMSF) were irradiated with an UV-B dose of ~200 J/m² for the indicated time points in the dark at 25°C. Tryptophan fluorescence spectra of control and UV-B irradiated protein samples were measured using excitation wavelength of 295 nm. The emission wavelengths were set in the range between 300 to 400 nm with the emission scan speed of 240 nm/min. The 0-h time point served as the control. (E) Normalized tryptophan fluorescence intensity per tryptophan residue in full length recombinant Pol λ, Del 1, Del 2 and Del 3 fragments under control condition (without UV-B exposure). (F) Normalized tryptophan fluorescence intensity per tryptophan residue in full length recombinant Pol λ, Del 1, Del 2 and Del 3 fragments after 4 h of UV-B (~200 J/m²) at room temperature.</p

UV-B mediated oxidative degradation of tryptophan residues to <i>N</i>-formylkynurenine in Pol λ and its N-terminal deletion mutants.

<p>(A) Normalized tryptophan fluorescence intensity per tryptophan residue in untreated control or (B) 4 h of UV-B exposed full length recombinant Pol λ, Del 1, Del 2 and Del 3 fragments. Fluorescence spectra were measured using excitation wavelength of 365 nm with the emission wavelengths ranging from 390 to 530 nm and emission scan speed of 240 nm/min, using 0.05 mg/mL of purified protein samples for analyzing the UV-B induced oxidation of tryptophan to <i>N</i>-formylkynurenine. (C) Tryptophan fluorescence spectra of 0.05 mg/ml of BSA (Sigma fraction V) under control or after 1 and 4 h of UV-B exposure. (D) Fluorescence spectra of control and UV-B BSA were measured using excitation wavelength of 365 nm with the emission wavelengths ranging from 390 to 530 nm to monitor the UV-B induced oxidation of tryptophan to <i>N</i>-formylkynurenine. (E) Normalized tryptophan fluorescence intensity per tryptophan residue in 4 h of UV-B exposed full length recombinant Pol λ and BSA. Fluorescence spectra were measured using excitation wavelength of 365 nm with the emission wavelengths ranging from 390 to 530 nm to compare the UV-B induced oxidation of tryptophan to <i>N</i>-formylkynurenine in Pol λ and BSA.</p

Parameters of equilibrium urea unfolding of recombinant full length and N terminus truncated forms of AtPol under control and after UV-B irradiation or high NaCl treatment.

<p># (Errors represent SD from triplicate measurements)</p><p>Parameters of equilibrium urea unfolding of recombinant full length and N terminus truncated forms of AtPol under control and after UV-B irradiation or high NaCl treatment.</p