InP Quantum Dots: Probing the Active Domain of Tau Peptide Using

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InP Quantum Dots: Probing the Active Domain of Tau Peptide Using Energy Transfer Shyamala Thirunavukkuarasu, Athira George, Anu Thomas, Anoop Thomas, Vinesh Vijayan, and K George Thomas J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b01533 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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The Journal of Physical Chemistry

InP Quantum Dots: Probing the Active Domain of Tau Peptide using Energy Transfer Shyamala Thirunavukkuarasu,* Athira George, Anu Thomas, Anoop Thomas, Vinesh Vijayan and K George Thomas* School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram (IISERTVM), Vithura, Thiruvananthapuram 695551, India E-mail: [email protected]; [email protected] AUTHOR INFORMATION Corresponding Authors *Email TS: [email protected] *Email KGT: [email protected] Current address Athira George: University of California, San Diego, United States. Anu Thomas: University of Texas, Southwestern Medical Centre, Dallas, United States. Anoop Thomas: University of Strasbourg, CNRS, ISIS and icFRC, Strasbourg, France.

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ABSTRACT Aggregation of Tau, a natively unfolded protein, is responsible for tauopathies, a class of neurodegenerative disorders. An active peptide sequence containing 20 amino acids is selected from the Tau microtubule binding region, which includes the essential V306-K311 residue, to monitor the structural change that initiates aggregation at very low concentrations. The synthesis of peptide sequence is accomplished by employing solid phase protocols. The active domain of Tau possesses an amino functionality on lysine and free thiol on cysteine. The former end is selectively labeled to rhodamine 101 which is further bound to InP/ZnS quantum dot surface through the thiol linkage. Efficient resonance energy transfer is observed in its unfolded conformation which is confirmed using various steady state fluorescence techniques. The average distance between the quantum dot core and the chromophore is probed by FRET as 24.5 ± 0.8 Å. Heparin, a negatively charged glycosaminoglycan, is used for inducing aggregation of the active domain of Tau peptide. Structural changes in the peptide monomer, on addition of heparin, could be monitored at nanomolar concentrations through the inhibition of energy transfer from quantum dots to rhodamine dye.

INTRODUCTION Neurodegenerative disorders such as Alzheimer’s disease and frontotemporal dementias are characterized by the presence of proteinaceous aggregates called neurofibrillary tangles and paired helical filaments (PHFs).1 A microtubule-associated protein, Tau is the major component of these aggregates. The structural features of Tau have been well established: (i) it is a natively unfolded protein with over 440 amino acids in the longest isoform, (ii) lacks hydrophobic amino acid moieties and (iii) highly soluble in biological conditions.2 The biological function of Tau protein is to promote and stabilize the assembly of tubulin monomers into microtubules. However, post-translational modifications such as hyperphosphorylation, induces the aggregation of Tau into extremely insoluble, protease resistant beta-sheet structures (PHFs) which causes various neurodegenerative disorders.1 The objective of the present investigation is to design a water-soluble donor–acceptor construct linked ACS Paragon Plus Environment

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together with the active peptide sequence from the Tau microtubule binding region and investigate the initial steps of aggregation by following energy transfer process. It is well established that the aggregation of Tau protein occurs primarily at the microtubule-binding domain containing three or four repeats. The hexapeptide motifs

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VQIINK281 from the second repeat

(R2) and 306VQIVYK311 from the third repeat (R3) of Tau are crucial in the conformational switch from its unfolded form to a beta-sheet structure.3 In full-length protein, the beta structure formed by a small portion of the sequence is difficult to detect by X-ray or other spectroscopic methods since it gets buried inside the surrounding random coil structure. Hence investigations are carried out by using its active domains, which reflect its native functionality and include the core region, that are usually found in PHFs. In the present study, a 20 amino acid peptide (305KVQIVYKPVDLSKVTSKCGS324) from Tau R3 domain (denoted as PP) that includes the essential V306-K311 residues is selected to investigate the aggregation in vitro. One of the convenient ways to induce aggregation is by adding polyanions (such as polysaccharides like heparin, polyglutamate peptide, nucleic acids such as RNA and fatty acids micelles like arachidonic acid) to the active sequence of Tau protein.1, 3 The extended negative charges in these inducers neutralize the positive charges in Tau sequence and promotes protein aggregation in a concentration dependent manner.1 A detailed solution-state NMR investigation, highlighting the structural importance of the hexapeptide sequence, and other residues in fibrillization of our chosen sequence has been reported recently by Vijayan and coworkers.4 Single molecule energy transfer that probes below the ensemble average was reported by Rhoades and coworkers to identify and characterize monomer conformational changes in an extended sequence from the R3 domain.5 The widely employed method to follow protein aggregation is the Thio T assay. It is based on the increase in fluorescence intensity of a benzothiazole salt on binding to an extended beta-sheet structure in fibrils.6 However, most of the commonly used techniques such as Thio T fluorescence, CD spectroscopy and other physical methods (for e.g., turbidity and sedimentation) are insensitive at lower concentrations and it becomes difficult to probe the initial stages of aggregation.7

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Understanding the structural changes, using steady state emission techniques, that lead to the initiation of aggregation at sub-micromolar concentrations are significantly important. Heparin is commonly used for inducing aggregation under laboratory conditions. The morphological characteristics of the PHFs observed in vivo are found to be similar to that seen during the in vitro polymerization of Tau, induced by heparin. Therefore, efforts in the treatment of neurodegenerative diseases include high throughput screening for active molecules that inhibit heparin-induced fibril formation of Tau.8 Although the presence of proteinaceous aggregates is considered as hallmark of neurodegenerative diseases, recent research points that even the soluble oligomeric forms of the protein are cytotoxic.6-7 The aggregation of protein in vitro involves nucleation and exponential growth, characterized by an initial lag phase. The long initial lag phase is analogous to the prolonged clinical phase observed in these neurodegenerative disorders. In this manuscript, we have used Förster resonance energy transfer (FRET) to characterize the active domain of Tau peptide during the initial step of aggregation at sub-micromolar concentrations. FRET has become a powerful tool as a “spectroscopic ruler” for measuring nanoscale separation between macroscopic systems.9-10 Use of quantum dots (QDs) as FRET donors/acceptors have several advantages, compared to conventional chromophoric dyes and their use for investigating energy transfer has been reviewed.11-18 Mattoussi and coworkers have linked a series of engineered peptides and proteins on CdSe/ZnS QDs and monitored the kinetics of self-assembly by following energy transfer between QDs and dye-labeled proteins/peptides.19 The use of FRET based QD sensors for the detection of (i) proteolysis20 (ii) specific and nonspecific DNA21 (iii) small molecules such as dopamine and monosaccharide22 (iv) hazardous chemicals such as TNT23 and (v) various enzyme activity assays14 have also been reported. Experimental investigations by Mulvaney and coworkers on the efficiency of energy transfer (ET) between CdSe QDs and covalently linked organic dyes have shown that a single chromophore can effectively quench the exciton luminescence of the nanocrystal.24 Resulting from an effective increase in the absorption cross-section of the dye upon conjugation, the relative quantum yield for energy transfer from the QD to the dye is as high as 90%. The group has also reported the insufficiency of the existing models for calculating the efficiency of energy transfer in nanoparticle−dye ACS Paragon Plus Environment

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conjugates using steady-state measurements.25 The authors have effectively adopted Poisson statistics for investigating the distance dependence of energy transfer in QD-chromophore conjugates.25 In the present study, a 20 amino acid peptide which forms the active domain of Tau responsible for various neurodegenerative disorders have been selectively labeled with the rhodamine 101 (denoted as Rh; Scheme 1) using the amino functionality on lysine and further assembled to InP/ZnS QDs (InP; Scheme 1) surface using the free thiol on the cysteine. By following FRET, the labeled Tau peptide residue namely the QD-peptide construct (Rh-PP-InP; Scheme 1) is characterized and the aggregation in the presence of an inducer (heparin) is investigated. EXPERIMENTAL SECTION Materials. All chemicals are of analytical grade, and high performance liquid chromatography (HPLC) or spectroscopic grade solvents are used without further purification. Nα-Fmoc (N-(9fluorenylmethoxycarbonyl) protected amino acids, dithiothreitol (DTT), heparin sodium salt from porcine intestinal mucosa, tetramethylammonium hydroxide (TMAOH), mercaptosuccinic acid (MSA), tris(hydroxymethyl)aminomethane (Tris base), boric acid, agarose, hexafluoroisoproponal (HFIP), trifluoroacetic acid (TFA), triisopropylsilane (TIS), N,N’-diisopropylcarbodiimide (DIPC), NMethylmorpholine (NMM), piperidine, acetic anhydride, indium acetate, myristic acid, sulphur, octadecene, trioctylphosphine, diethylzinc solution (1M in hexane), rhodamine101 inner salt, quinine sulphate and dialysis tubing cellulose membrane molecular weight cut-off (MWCO) 14,000 Da are purchased from Sigma Aldrich. Fmoc rink amide MBHA (4-methylbenzhydrylamine) resin and N,N,N,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU) are purchased from Merck. Tris(trimethylsilyl)phosphine is purchased Across Organics. Dimethylformamide, diethylether, methanol, chloroform, acetonitrile are purchased from Spectrochem (India). Milli-Q-water (Millipore) is used for preparation of all aqueous solutions.


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Phase transfer of InP quantum dots to aqueous solution Myristic acid capped InP nanocrystals overcoated with ZnS (InP) are synthesized using a previously reported procedure.26 The core-shell QDs are purified by precipitation using a mixture (1:4) of methanol and acetone followed by centrifugation. The hydrophobic QDs are rendered water-soluble adopting a ligand exchange procedure using MSA and tetramethylammonium hydroxide at pH 11 (Figure 1A). The hydrophilic QDs is purified by precipitation, redispersed in water and further dialyzed using MWCO membrane 14,000 Da. The yield of InP/ZnS QDs transferred from toluene to aqueous medium using MSA as ligand is ~70%, which is estimated by following a method reported by Reiss and coworkers.27 The pH of the MSA capped InP is 7.2 in water. The concentration of place exchanged QDs is estimated using a literature procedure.28 InP possess inherently low quantum efficiency (vide infra)29: in the present case the relative quantum yield of InP in toluene and water are estimated as 4% and 0.4% using quinine sulphate as standard. Synthesis, labeling and purification of the peptide The twenty amino acid peptide sequence, (KVQIVYKPVDLSKVTSKCGS) from the R3 domain of Tau protein is synthesized using an automated solid phase peptide synthesizer under constant nitrogen flow conditions. The Nα-Fmoc amino acids are used with the following side-chain protecting groups: Boc for lysine, Trt for glutamine and cysteine, t-Bu for tyrosine, serine and threonine, O t-Bu for aspartic acid (Boc: tert-butoxycarbonyl; Trt: trityl; t-Bu: tertiary butyl). The N-terminal acetylated or non-acetylated peptide (0.2 mmol) with a C-terminal amide is assembled on Fmoc Rink amide MBHA resin (0.38 mmol/g). The addition of each amino acid to peptide sequence (reagent/time) is programmed in synthesizer as three steps: (i) deprotection using 20% piperdine in DMF (2 x 15 min), (ii) activation of the amino acid using HBTU (0.18 mmol) and NMM (0.4 M) dissolved DMF (1x 30 s) (iii) coupling (1x 20 min). After the completion of the required peptide sequence, the resin is washed with DCM and dried under vacuum. Dye labeling of peptide: Labeling is performed manually with the non-acetylated peptide after the deprotection of the final N-terminal group and before cleaving from the resin (Figure 2A). Dried resin ACS Paragon Plus Environment

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(50 mg) containing non-acetylated peptide is suspended in DMF (5 mL) and allowed to swell for 30 min. A two fold molar excess of the rhodamine 101(Rh), HBTU and DIPC, dissolved in 100 µL of DMF, is added to the resin slurry. The reaction is stirred for 3 h. The resin is washed sequentially with DCM, DMF and a mixture of methanol/DCM to remove the excess dye. The labeled resin is allowed to dry in vacuum. Peptide cleavage protocol: The acetylated peptide (Ac-PP) and rhodamine labeled peptide (Rh-PP) are cleaved from the resin using the cocktail consisting of 88% TFA, 5% water, 2% TIS and 5% DTT. A scheme illustrating the synthesis of Ac-PP and Rh-PP are presented in Figure 2A. The cleavage is performed for the first 20 min on ice and further at 22 °C with continuous stirring under nitrogen atmosphere for not exceeding 2 h. The peptide-resin solution is filtered and concentrated under a nitrogen stream. The cleaved peptide is isolated by precipitation of the filtrate in cold diethyl ether. The precipitate is removed from the supernatant by centrifugation at 5000 rpm for 7 min. The peptide is vacuum dried and lyophilized with glacial acetic acid. Reverse-Phase HPLC purification of peptides: The purifications of lyophilized Ac-PP and Rh-PP are performed using a Shimadzu prominence liquid chromatograph with a Phenomenex luna 5u C8 column. Buffer A (0.1% TFA in water) and buffer B (0.1% TFA in acetonitrile) with a flow rate of 1 mL/ min and an eluent gradient from 5% to 95% acetonitrile is used. The peptides are dissolved in small amount of buffer A and treated with HFIP prior to injection. Appropriate fractions is collected by monitoring the absorbance at 214 nm and 280 nm for Ac-PP and also at the dye absorption maxima 575 nm for Rh-PP (Figure S1, Supporting information). Both the peptides, Ac-PP and Rh-PP, are eluted at 55% acetonitrile gradient. The collected fractions are characterized by matrix assisted laser desorption timeof-flight mass spectrometry (Figure S2). Both peptides are lyophilized thrice from deionised water and stored at -20 °C.


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Steady-state and time-resolved measurements All photophysical experiments are carried out in micro-volume quartz cuvette (1.4 mL, Starna USA) of path length 1 cm. Absorption spectral studies are carried out using a UV-vis-NIR spectrophotometer (Shimadzu UV-3600). Emission and excitation measurements are performed using spectrofluorimeter (Horiba Jobin Yvon – Fluorolog 3) with slit width 3/3 nm. Circular dichroism spectra are recorded using CD spectrometer (JASCO J815). Emission lifetimes are measured using a picosecond time-correlated single photon counting system (Horiba Jobin Yvon-IBH). Solutions are excited at 405 nm using a pulsed diode laser (Nano LED-405L;

InP Quantum Dots: Probing the Active Domain of Tau Peptide Using

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