Site-Specific Fluorescence Depolarization Kinetics Distinguishes the

Aug 25, 2017 - To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took...
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Site-Specific Fluorescence Depolarization Kinetics Distinguishes the Amyloid Folds Responsible for Distinct Yeast Prion Strains Dominic Narang,†,‡,§,∥ Hema M. Swasthi,†,§,⊥ Sayanta Mahapatra,†,‡ and Samrat Mukhopadhyay*,†,‡,⊥ †

Centre for Protein Science, Design and Engineering, ‡Department of Biological Sciences, and ⊥Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Sector 81, Knowledge City, S.A.S. Nagar, Mohali 140306, Punjab, India S Supporting Information *

ABSTRACT: The prion determinant of a yeast prion protein, Sup35NM, assembles into β-rich amyloid fibrils that switch the nonprion [psi−] state to the prion [PSI+] state of yeast. Previous studies showed that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4 and 37 °C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI+] phenotype, whereas Sc37 shows a weak phenotype. To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took advantage of the nonoccurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed a conformational mobility map to be constructed. Nearly all of the residue positions demonstrated higher local flexibility in Sc4 amyloid, which exhibits a strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain than in the central region. The M-domain is partially exposed and exhibits a higher amplitude of local mobility, indicating a lower degree of chain packing in the amyloid state, as well as a higher mobility in the Sc4 state compared to the Sc37 state. The altered local conformational dynamics in these two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance patterns of strong and weak prion strains.

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INTRODUCTION Prions are self-propagating protein aggregates that are capable of displaying protein-based inheritance.1−3 An intriguing feature of prions is the existence of multiple, related conformations in the amyloid state. These amyloid forms, comprising distinct supramolecular structures, are known as prion strains; they are faithfully transmitted and demonstrate a variety of biological phenotypes.4,5 The earliest evidence of amyloid polymorphism came from a study of the mouse prion protein in which the scrapie forms isolated from different mice showed variations in the protease-resistant cores.6,7 In addition to prions, several other protein amyloids associated with neurodegenerative diseases are known to exhibit polymorphism and strain diversity.8 Therefore, the strain phenomenon appears to be quite prevalent in various prion disorders and deadly neurodegenerative diseases. However, fungal prions are functional amyloids that are beneficial to yeast cells and serve as an outstanding model system for studying many unusual properties of prions, such as the strain phenomenon, in addition to deciphering the mechanisms of prion propagation and transmission. The propagation of [PSI+] prion in yeast (Saccharomyces cerevisiae) has been well-studied and occurs by the conversion of soluble functional Sup35p, a translation termination factor, into an inactive amyloid state.9,10 Sup35 © XXXX American Chemical Society

protein consists of three domains: N-terminal (N, residues 1− 123), middle (M, residues 124−253), and C-terminal (C, residues 254−685). The Q/N-rich N-domain is responsible for both prion induction and maintenance, and the positively charged M-domain is involved in maintaining the solubility of the on-prion form [psi−] of Sup35 (Figure 1A).11,12 It has been shown that the NM-domain, denoted as Sup35NM, is sufficient for prion-based inheritance, whereas the C-domain is essential for translation termination activity.12 Additionally, it has been documented that chaperones such as Hsp104, Hsp70, and Hsp40 play an important role in the propagation and inheritance of the [PSI+] prion.2,13,14 Sup35NM adopts an intrinsically disordered state in its native monomeric form and aggregates into a self-propagating β-rich amyloid state that is responsible for the non-Mendelian inheritance trait in yeast.15,16 Previous studies demonstrated that two distinct forms of amyloid fibrils derived from Sup35NM of Saccharomyces cerevisiae can be generated in vitro at two different temperatures, namely, 4 and 37 °C, which are designated as Sc4 and Sc37, respectively.17−19 A careful Received: June 6, 2017 Revised: August 20, 2017 Published: August 25, 2017 A

DOI: 10.1021/acs.jpcb.7b05550 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. (A) Amino acid sequence of Sup35NM. Single Trp mutation sites are indicated in red. The putative boundary between the N- and Mdomains is also shown. (B) Thioflavin-T (ThT) fluorescence kinetics of wild-type and single-Trp mutants of Sup35NM during amyloid formation, showing nearly identical assembly kinetics at room temperature. The protein concentration was 2.5 μM. The reaction mixtures were stirred at 100 rpm using magnetic beads in quartz cuvettes, and the fluorescence intensity was measured at regular intervals. Representative individual kinetics traces are shown in Figure S1. (C,D) AFM images of Sup35NM fibrils formed at (C) 4 and (D) 37 °C with a height of ∼5 nm. (E) Trp emission maxima of different residue positions in two different amyloid forms, Sc4 and Sc37 (both N- and M-domains are also shown). The excitation and emission slit widths were 0.8 and 4 nm, respectively.

register organization. These structural features responsible for prion stain diversity can involve variations in the lengths of the β-rich amyloid cores, locations of the folds, and lengths of the connecting loops, as well as differences in the staggering of βstrands.18,27,28 However, detailed molecular insights into the chain organization and dynamics within distinct strains at the residue-specific level still remain elusive. In this work, we have studied the local conformational mobilities in two distinct amyloid states that are responsible for displaying prion strain diversity. To discern the residue-specific structural attributes associated with the distinct amyloid folds, we took advantage of the fact that Sup35NM is devoid of tryptophan (Trp) and introduced single Trp residues at 18 different locations encompassing the entire polypeptide length. We then employed highly sensitive picosecond time-resolved fluorescence spectroscopy measurements to construct conformational mobility maps of the polypeptide chain in the two amyloid forms.

characterization of Sc4 and Sc37 prions revealed that they are composed of distinct conformational states and give rise to diverse strains.17 These studies also revealed that the amyloid core of Sc4 fibrils is shorter and less stable than that of Sc37 fibrils.18,19 Interestingly, when these recombinantly generated amyloids are introduced into yeast cells, Sc4 fibrils exhibit a strong [PSI+] phenotype, whereas Sc37 fibrils show a weak [PSI+] phenotype. This phenotypic role reversal under in vitro and in vivo conditions was explained on the basis of the mechanical stability of the fibrils. It was conjectured that Sc4 amyloid, being mechanically less stable, is efficiently fragmented by the chaperone machinery, which provides new seeds for the autocatalytic conversion and propagation of the soluble Sup35 into prions, giving rise to a strong [PSI+] phenotype. On the contrary, Sc37, being structurally more robust, is less prone to chaperone-mediated disintegration, which eliminates further polymerization of soluble Sup35, giving rise to a weak [PSI+] phenotype.19 A variety of structural models of the protein organization within Sup35NM fibrils have been proposed. For instance, the parallel in-register structural model suggests that the amyloid assembly is primarily governed by intermolecular interactions whereby identical residues of different Sup35NM protein molecules are aligned and stacked on top of each other.20−24 On the contrary, the β-helix model25 suggests that the interplay of both intra- and intermolecular contacts is essential for prion formation.26 According to this model, intermolecular contacts are formed between the head (top) and tail (bottom) regions of adjacent Sup35NM proteins, whereas intramolecular contacts prevail in the central core region.26 Recent studies have provided more compelling evidence in favor of parallel inregister packing.27−30 A growing body of evidence has indicated that the presence of prion strain variants is a result of altered structural attributes in multilayered and cross-β parallel in-

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EXPERIMENTAL SECTION Materials. Guanidine hydrochloride (GdmCl), tris(hydroxymethyl) aminomethane (Tris), sodium chloride, sodium phosphate monobasic dihydrate, sodium phosphate dibasic dihydrate, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), thioflavin T (ThT), and urea were purchased from Sigma (St. Louis, MO) and used without any additional purification. Isopropyl-β-thiogalactopyranoside (IPTG) and antibiotics (ampicillin, tetracycline, and chloramphenicol) were obtained from Gold Biotechnology (St. Louis, MO). Ni-NTA resin and Q-Sepharose were obtained from GE Healthcare Life Sciences (Wauwatosa, WI). Cloning and Mutagenesis. A DNA fragment encoding Sup35NM was amplified from yeast genomic DNA using

B

DOI: 10.1021/acs.jpcb.7b05550 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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using a picosecond rhodamine 6G dye laser (Spectra Physics, Mountain View, CA) coupled to a time-correlated singlephoton-counting setup, as described previously.31 Briefly, a mode-locked frequency-doubled Nd:YAG laser (Millennia X, Spectra Physics) was used to pump the dye laser, and the generated laser pulses were frequency-doubled to 295 nm. The full width at half-maximum of the instrument response function was ∼60 ps. For time-resolved intensity and anisotropy decay measurements, the emission monochromator was fixed at 345 nm with a bandpass of 8 nm, and the emission was collected using a microchannel plate photomultiplier (model 2809u, Hamamatsu Corp.). The fluorescence intensity decays were collected at the magic angle (54.7°) with respect to the excitation polarizer, and the peak count was ∼20000. For fluorescence depolarization kinetics, emission was collected at 0° (parallel) and 90° (perpendicular) with respect to the excitation polarizer. The perpendicular component of the fluorescence intensity was always corrected using the G-factor, which was collected independently. The anisotropy decays were analyzed by globally fitting I∥(t) and I⊥(t), and the anisotropy decays were fitted globally using a biexponential decay model that allowed the fast and slow rotational correlation times to be recovered as follows31

appropriate primers and cloned into the pET-23a expression vector. Single Trp residues were introduced at different positions using overlap extension polymerase chain reactions. The primers for single-Trp mutations are listed in Table S1. Protein Expression and Purification. C-terminal hexahistidine recombinant Sup35NM proteins were overexpressed in BL21 (DE3)/pLysS cells using 0.4 mM IPTG at 37 °C for 2 h. Harvested cells suspended in lysis buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0) were incubated in boiling water for 10 min. The boiled lysate was centrifuged at 11500 rpm for 30 min to remove the cell debris. The supernatant was precipitated using an equal volume of a saturated solution of ammonium sulfate at 4 °C. The pellet formed was dissolved in a buffer containing 8 M GdmCl and 20 mM phosphate buffer (pH 8.0) and kept at 4 °C overnight. The denatured protein solution was centrifuged to remove any insoluble particles, and the supernatant was subjected to Ni-NTA purification. After NiNTA purification, the protein was further purified on a QSepharose column. The purified protein was concentrated and precipitated using methanol. The protein pellet was resuspended in 70% methanol and kept at −80 °C until further use. Amyloid Fibril Formation. The precipitated protein was dissolved in 8 M urea and 20 mM phosphate buffer (pH 8.0) for 3 h at room temperature to monomerize. The denatured protein was passed through a 100-kDa filter (Millipore) to remove any pre-existing aggregates and then concentrated using a 3-kDa filter (Millipore) before the aggregation reaction. The concentrated protein was further centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was diluted in the aggregation buffer (5 mM Na2PO4, 150 mM NaCl, 5 μM ThT, pH 7.4). For ThT-binding experiments, the final concentrations of protein and urea were 2.5 μM and ∼80 mM, respectively. The reaction was carried out at room temperature under stirring at 100 rpm using magnetic beads. The time-dependent ThT fluorescence was monitored at room temperature by exciting at 450 nm, and the fluorescence emission was collected at 480 nm. For steady-state and timeresolved Trp fluorescence experiments on matured amyloid fibrils, fibril formation was carried out by performing a 100-fold dilution of the protein stock into aggregation buffer (5 mM phosphate, 150 NaCl, pH 7.4) that had been pre-equilibrated at 4 or 37 °C. The final protein concentration was 5 μM. The reaction was carried out by slow overhead rotation (10 rpm) for 24 h at 4 or 37 °C. After the fibrillation reaction, the solution was passed through a 50-kDa filter to remove any monomeric/lower-order species, and the concentrate was used for fluorescence measurements. Steady-State Fluorescence Measurements. Steady-state fluorescence measurements were performed on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ). For Trp fluorescence spectra, the excitation wavelength was set at 295 nm, and the excitation and emission slit widths were 0.8 and 4 nm, respectively. Fluorescence anisotropy measurements were performed by setting the excitation wavelength (λex) at 295 nm (bandpass = 1.5 nm) and the emission wavelength (λem) at 345 nm (bandpass = 10 nm) with an integration time of 0.5 s. All of the measurements were made at 24 ± 1 °C. The steady-state fluorescence anisotropy was estimated using the parallel (I∥) and perpendicular (I⊥) intensities and the G-factor as follows r = (I − I⊥G)/(I + 2I⊥G)

r(t ) = r0[βfast exp( −t /ϕfast) + βslow exp( −t /ϕslow )]

(2)

where r0 is the intrinsic fluorescence anisotropy; ϕfast and ϕslow are the short and long rotational correlation times, respectively; and βfast and βslow are the amplitudes associated with the short and long correlation times, respectively. The details of our global analysis are described in our previous publication.31 The values of βfast were used to construct the conformational mobility map. All of the measurements were performed at 24 ± 1 °C. Atomic Force Microscopy (AFM) Imaging. The aggregation reactions were performed as described above. An aliquot was withdrawn from the reaction mixture and deposited onto freshly cleaved mica (muscovite-grade V-4 mica from SPI, West Chester, PA). After incubation for 5 min, the mica was washed with water and dried under a nitrogen stream. Tappingmode AFM was performed using Bruker Innova atomic force microscope. The images were analyzed using WSxM 5.0 software.32

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RESULTS To monitor the differences in the structure and organization of the polypeptide chains within the two distinct amyloid forms, we employed site-specific and multiparametric fluorescence spectroscopy. For these experiments, we created 18 single-Trp mutants that were distributed over the entire NM polypeptide chain (Figure 1A). These residue positions were 2, 7, 21, 25, 31, 51, 58, 68, 86, 96, 104, 117, 121, 129, 137, 184, 221, and 250. The residue positions were chosen based on the fact that the mutations at these positions do not alter the prion-forming behavior of Sup35NM, as described previously.26 Additionally, we independently established by means of an aggregation assay of recombinantly expressed wild-type and single-Trp mutants of Sup35NM that the mutants indeed retained the aggregation behavior similar to that of the wild-type (Figures 1B and S1). The thermal stability data of the wild-type and a few mutants are shown in Figure S2. AFM imaging revealed that the fibrils formed at both 4 and 37 °C (Sc4 and Sc37) have similar overall nanoscale morphologies (Figure 1C). The heights of these

(1)

Time-Resolved Fluorescence Measurements. Timeresolved fluorescence decay experiments were carried out C

DOI: 10.1021/acs.jpcb.7b05550 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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Figure 2. (A) Steady-state fluorescence anisotropies of different residue positions in two amyloid states (Sc4 and Sc37). The standard deviations were estimated from three independent experiments. (B) Time-resolved fluorescence anisotropy decays [r(t)] and fits of single-Trp variants in Sc37 amyloid showing the contributions of fast (local) motion and slow dynamics. r0 is the time-zero anisotropy.

fibrils ranged between 4 and 6 nm. Next, to decipher the sitespecific structural information within the polymorphic strains, we monitored the changes in the tryptophan emission spectra of the monomeric and amyloid states (Sc4 and Sc37) of Sup35NM. In the denatured monomeric state, Trp fluorescence emission spectra of all of the residue positions showed a peak at ∼345 nm, suggesting that all of the residue positions are solvent-exposed (Figure S3). Upon conversion into the amyloid fibrils, almost all of the residue positions showed a blue shift compared to the unfolded monomeric form. However, careful scrutiny of the residue-specific emission maxima revealed that the extent of the blue shift varied within the respective Sc4 and Sc37 fibrillar states, as well as between these states (Figure S3). For instance, the residues at positions 21, 25, and 31 in the Ndomain showed the largest extents of blue shift in the Trp fluorescence spectra in both the Sc4 and Sc37 states, indicating that these positions are well-protected from bulk water compared to their locations in monomeric Sup35NM, which is consistent with previous studies.26 However, careful inspection revealed that these residues (residing at the head region of the N-domain) as well as the residue at 117 (tail region of the N-domain) are more buried in the Sc37 fibrils than in the Sc4 fibrils. On the contrary, the other residues in the N-domain are partially solvent-exposed in both states (Figure 1D). In contrast to the residues in the N-domain, the Trp residues located at positions 184, 221, and 250 within the Mdomain exhibited a moderate extent of blue shift in the emission maxima, indicating that the M-domain is somewhat exposed to water in the fibrillar states of both Sc4 and Sc37. Here again, a comparison between the emission maxima of the M-domain residues in Sc4 and Sc37 revealed that nearly all of the residues in Sc37 are relatively less solvent-exposed than those in Sc4, similarly to the case observed for the N-domain. Taken together, these observations support the conclusion that the head and tail regions of the N-domain actively participate in the formation of the (solvent-excluded) dry interface of the amyloid core. Next, we carried out steady-state fluorescence anisotropy measurements, which reported the overall rotational flexibility of Trp located at various residue positions in both the monomeric form and the amyloid variants Sc4 and Sc37 (Figure 2A). The monomeric unfolded form showed a low anisotropy (∼0.05) at all locations, whereas the aggregated state exhibited anisotropy value ranging from 0.1 to 0.2 depending on the location. It was observed that the N-domains in both Sc4 and Sc37 exhibited high anisotropies, indicating that the N-domain residues get recruited within the amyloid core. A finer comparison revealed that (a) the residues

spanning positions 2−31 (head region) and 96−117 (tail region) in the N-domain showed slightly higher anisotropy than the residues at positions 51−86, suggesting higher conformational constraints at the two terminal segments of the N-domain, and (b) nearly all of the residues in the Sc37 fibrils demonstrated more rigidity and, hence, more structural ordering than those of the Sc4 fibrils, in accordance with our tryptophan emission results. On the other hand, the M-domain showed lower anisotropy than the head and tail regions of the N-domain, suggesting a higher mobility of the M-domain segment. We also point out that the C-terminal segment of the M-domain is more structured in Sc37 than in Sc4, indicating a longer amyloid core in the Sc37 fibrils. Hence, our steady-state fluorescence emission and anisotropy experiments revealed that the Sc37 fibrils comprise a more extended core and are more structured and more solvent-protected than the Sc4 fibrils. Because the steady-state fluorescence anisotropy readout is highly convoluted as a result of the changes in the fluorescence lifetimes and the presence of multiple rotational correlation times, we next carried out time-resolved fluorescence anisotropy measurements to delineate the site-specific conformational mobilities. To directly capture the dynamical signatures of these two amyloid states, we carried out fluorescence depolarization kinetics experiments.31,33−35 Fluorescence depolarization kinetics of Trp monitored by the time-resolved anisotropy decays [r(t)] of various residue positions allows the components of the local and the global motions on the picosecond to nanosecond time scales to be separated in a site-specific manner.35 The depolarization kinetics of various Trp positions can be described by biexponential decay functions containing two rotational correlation time components: The fast component (ϕfast ≤ 0.2 ns) corresponds to the local motion of the fluorophore, whereas the slow component (ϕslow) represents the global tumbling of the macromolecule (cf. eq 2). For amyloid fibrils, the time-dependent anisotropy [r(t)] does not decay to zero within the time scale of fluorescence emission, and therefore, they exhibit a (limiting) residual anisotropy (r∞) because of a much greater value of ϕslow due to the large aggregate size. However, the amplitudes of the fast rotational correlation times (βfast) at various residue positions serve as a valuable readout of local mobility.35 The differences in the r∞ values of different residue positions arise from different extents of (fast) local depolarization. Figure 2B shows the fluorescence depolarization kinetics of different Trp locations in the Sc37 amyloid state. N-terminal residue 7 exhibited very little local flexibility (