Structural Characterization of Nanoscale Meshworks within a

Article pubs.acs.org/Biomac

Structural Characterization of Nanoscale Meshworks within a Nucleoporin FG Hydrogel Marcel Petri,† Steffen Frey,‡ Andreas Menzel,§ Dirk Görlich,‡ and Simone Techert*,† †

Research Group of Structural Dynamics of (Bio)chemical Systems and ‡Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany § Paul Scherrer Institut, 5232 Villigen PSI, Switzerland S Supporting Information *

ABSTRACT: The permeability barrier of nuclear pore complexes (NPCs) controls all exchange of macromolecules between the cytoplasm and the cell nucleus. It consists of phenylalanine−glycine (FG) repeat domains apparently organized as an FG hydrogel. It has previously been demonstrated that an FG hydrogel derived from the yeast nucleoporin Nsp1p reproduces the selectivity of authentic NPCs. Here we combined time-resolved optical spectroscopy and X-ray scattering techniques to characterize such a gel. The data suggest a hierarchy of structures that form during gelation at the expense of unstructured elements. On the largest scale, protein-rich domains with a correlation length of ∼16.5 nm are evident. On a smaller length scale, aqueous channels with an average diameter of ∼3 nm have been found, which possibly represent the physical structures accounting for the passive sieving effect of nuclear pores. The protein-rich domains contain characteristic β-structures with typical inter-β-strand and interβ-sheet distances of 1.3 and 0.47 nm, respectively. During gelation, the formation of oligomeric associates is accompanied by the transfer of phenylalanines into a hydrophobic microenvironment, supporting the view that this process is driven by a hydrophobic collapse.

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INTRODUCTION Eukaryotic cells are subdivided into two major compartments, the cytoplasm and the nucleus, which are equipped with complementary biosynthetic machineries. Because nuclei lack the ability to synthesize proteins, they rely on the supply with proteins synthesized in the cytoplasm. Conversely, ribosomes, tRNAs, and mRNAs needed in the cytoplasm are exclusively produced in the nucleus. This biosynthetic division of labor therefore necessitates a steady exchange of macromolecular products between both compartments.1,2 The nuclear envelope embraces the nucleus and poses a physical barrier that restricts macromolecular exchange between the nucleus and cytoplasm to nuclear pore complexes (NPCs). NPCs are guarded by a permeability barrier of remarkable selectivity: metabolites and small proteins can diffuse through the barrier, whereas passage of macromolecules exceeding a diameter of ∼5 nm is efficiently suppressed.3 Macromolecules larger than this exclusion limit can efficiently pass the barrier only when bound to nuclear transport receptors (NTRs) serving as shuttles. Importantly, NTRs and their cargo complexes penetrate the barrier at high rate without leaving long-lived perforations. This suggests that the barrier tightly seals around the translocating species.3,4 NPCs are built up from multiple copies of ∼30 different proteins called nucleoporins. A critical subset contains so-called “FG repeat domains” with up to 50 motives of the amino acid sequence FG, FxFG, or GLFG (single-letter amino acid code). © 2012 American Chemical Society

FG repeat domains are assumed to be intrinsically disordered;5,6 that is, they have only little well-defined secondary structure. Interactions between NTRs and FG motives are crucial for facilitated NPC passage of NTR•cargo complexes.7,8 Understanding the permeability barrier and the role of FG repeats in its formation remains challenging, and various models have been suggested.1,9−11 The “selective phase model”, for instance, suggests that FG repeats of various nucleoporins interact with each other, thereby forming a sieve-like 3-D hydrogel within the central channel of the NPC.4,12,13 According to this model, the mesh size determines the passive size-exclusion limit of the barrier. The selective phase model is supported by the observation that FG repeat domains of several yeast nucleoporins form elastic hydrogels with fascinating characteristics under native conditions.4,12,14 These gels exhibit permeability and sorption properties comparable to those observed for authentic NPCs: They allow an up to 20.000-fold faster entry of a large NTR•cargo complex compared with the cargo alone.4,12 The selective phase model can explain this striking effect because FG motifs not only bind NTRs7,8,15 but also are essential for gel-formation14 and hence for creating inter-repeat contacts. Binding of an NTR to an FG motif might therefore interrupt Received: March 16, 2012 Revised: April 26, 2012 Published: May 9, 2012 1882

dx.doi.org/10.1021/bm300412q | Biomacromolecules 2012, 13, 1882−1889

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degree polynomial, and the peak positions were determined by calculating the second derivative of the underlying spectra.19 Fluorescence Spectroscopy. Fluorescence spectra of Nsp12−601 were measured with a Fluorolog 3−22 (JOBIN YVON-SPEX, Munich, Germany) in 1 mm path-length suprasil quartz cuvettes (Hellma, Müllheim, Germany). For the emission experiments, spectra were taken in 0.5 nm steps at a fixed excitation wavelength of 252 nm. Slits were adjusted to 5 and 1 nm bandwidth for excitation and emission, respectively. Automatic correction for the wavelength dependence of the lamp intensity, monochromator transmission, and photomultiplier response was performed. Temperature-dependent experiments were executed using the static exchange gas continuous-flow cryostat Optistat Cf (Oxford Instruments NanoScience, Abingdon, England). Circular Dichroism Spectroscopy. Time-resolved CD spectra were measured on a Chirascan spectropolarimeter (Applied Photophysics, Leatherhead, United Kingdom) in 10 μm path-length suprasil quartz cuvettes (Hellma, Müllheim, Germany). Spectra were obtained from 190 to 350 nm with a 0.4 nm step and 1 nm bandwidth. Because of a high absorbance below 200 nm, the concentration of Nsp12−601 had to be limited to 30 mg/mL. As a consequence, the kinetics derived from CD spectroscopy may only be compared qualitatively to measurements of higher concentrations. To derive quantitative information on the secondary structure content, we evaluated the far-UV CD data in intermediate stages of hydrogel formation by the commonly used programs CDSSTR, CONTIN/LL, and Selcon3.20 Because of the best correlation between experimental data and fit, we used CDSSTR for an overall determination of the secondary structure content during the hydrogel formation process. The basis set SP22X, which covered the largest wavelength range of 178−260 nm, served as reference data set. Fourier Transform Infrared Spectroscopy. FTIR spectra were collected on a Perkin-Elmer Spectrum BX II (Perkin-Elmer, Waltham, MA) spectrometer with the solution pressed between calcium fluoride plates. A reference spectrum was recorded in advance and subtracted from the data. Because of the strong absorption of water between 1640 and 1560 cm−1, the usage of D2O was required in the amide I (1700− 1600 cm−1, CO stretching) region.21 Samples were prepared in potassium phosphate buffer yielding a final concentration of 200 mg/ mL protein at neutral pH. Time-resolved IR spectra were collected from 2000 cm−1 (5000 nm) to 1000 cm−1 (10 000 nm), taking four frames at a resolution of 1 cm−1 and interval of 0.5 cm−1. The temporal resolution of the experiment was set to 1 min. For an investigation of N−H stretching vibrations (3500−3200 cm−1), samples were prepared analogously but in H2O. Spectra were scanned eight times in a range from 4000 cm−1 (2500 nm) to 600 cm−1 (16 667 nm). Step width and energy resolution were set to 1 and 0.5 cm−1, respectively. Wide-Angle X-ray Scattering. X-ray diffraction patterns were recorded on a custom-made wide-angle X-ray scattering (WAXS) device. In brief, it comprised a modular X-ray source, iMOXS (IFG Institute for Scientific Instruments, Berlin, Germany), a one-circle goniometer (Huber, Rimsting, Germany), and a CCD camera, XRa-PISCX (Princeton Instruments, Trenton, NJ) to collect images. X-rays were monochromatized (λ = 1.54 Å) by a Ni-filter and focused on the sample by means of a montel multilayer optic, Quazar ELM21 (Incoatec, Geesthacht, Germany). The system was calibrated using silver behenate. The protein was prepared as stated above and pipetted in an O-ring placed on kapton foil. Then, it was covered with kapton foil to prevent evaporation of water during gel formation. After 2.5 h, both foils were removed, and the sample was mounted in the goniometer. Small-Angle X-ray Scattering. Small-Angle X-ray Scattering (SAXS) data were collected at the cSAXS beamline (X12SA) of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland). The sample-to-detector distance was 7.15 m and the selected X-ray wavelength was 1.1 Å. Samples were placed in glass capillaries of 1 mm in diameter (Hilgenberg, Malsfeld, Germany), and images were taken with 100 ms exposure time at an interval of 5 min by the Pilatus 2 M detector. Radiation damage has been avoided by moving the capillary in horizontal direction with respect to the incident X-rays, thereby probing a different sample volume with each run.

inter-repeat contacts, open the corresponding mesh, and allow the NTR to pass. The observation that the FG hydrogel is stable even at temperatures up to 95 °C supports the view that hydrophobic interactions are a critical element of the gel.14 FG repeat-derived hydrogels are of potential relevance not only for understanding the function of the nuclear pore but also for the development of structures with defined internal spaces as, for example, needed for drug storage.16 With respect to this, especially the hydrogel formed by the FG repeat domain (amino acids 2 to 601) of the yeast nucleoporin Nsp1p (Nsp12−601) could in general serve as a paradigm for FG hydrogels, as it is easily accessible for in vitro experiments. The ability of FG repeat hydrogels to sort proteins and their complexes in native form at high precision and rate might render these materials applicable for biomolecular filtration and separation techniques. A mechanistic understanding of the barrier’s selectivity requires a comprehensive insight into the structure and dynamics of such FG hydrogels. The amorphous nature of FG hydrogels makes them, however, refractory to analysis by Xray crystallography. The only detailed structural description available thus far relied therefore on solid-state NMR (ssNMR).17 This study provided evidence of the existence of interchain β-sheets within the Nsp1 FG hydrogel, but the structural organization of such gel at larger length scales remained elusive. Limited NMR spectral resolution made it also impossible to probe interactions between the critical Phe residues that are essential for gelation and NTR-binding. Here we report on a time-resolved optical spectroscopic and X-ray scattering characterization of Nsp1 FG repeat hydrogel formation on the nanoscale. The spectroscopic techniques revealed a transfer of Phe side chains into a hydrophobic environment and conformational changes of the protein backbone during gelation. Complementary X-ray scattering methods allowed determining details of the β-sheets and the structural organization on larger scales. It appears as if compact, protein-rich microphases alternate with microphases of lower local protein concentration. We suggest that these protein-poor microphases are the preferred pathways for passage of inert material and discuss possible pathways for NTRs and their cargo complexes.

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MATERIALS AND METHODS

Preparation of FG Repeat Hydrogels. Untagged Nsp1 repeat domain (Nsp12−601) was prepared exactly as previously described.18 Hydrogels were prepared by dissolving Nsp12−601 (TFA salt) in 200 mM Tris-HCl (pH 8.5) at a final concentration of 200 mg/mL. The resulting solution displayed a neutral pH (7.3−7.5). Hydrogel formation was allowed to proceed at room temperature. Hydrogels of shorter fragments (Nsp12−175 and Nsp1176−601) of the Nsp1 repeat domain were prepared analogously. For circular dichroism (CD) experiments, lyophilized Nsp12−601 was dissolved in potassium phosphate buffer to yield a final concentration of 30 mg/mL protein, 130 mM phosphate, and a neutral pH. Absorption Spectroscopy. Absorption spectra were recorded using a Cary-5E UV−VIS-NIR spectrophotometer (Varian Australia) in 1 mm path-length suprasil quartz cuvettes (Hellma, Müllheim, Germany). Absorption spectra characterizing the solution and gel state of Nsp12−601 were recorded from 200 to 400 nm in 0.5 nm steps and 0.2 s averaging time. Each spectrum was automatically corrected by use of a reference cuvette containing buffer. For time-resolved secondderivative absorption spectroscopy, spectra were obtained from 248 to 273 nm with 0.02 nm step width and 0.1 s collection time per step. To get a better wavelength resolution, the spectra were fitted by a sixth 1883

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The scattering intensities I(q) obtained were circularly averaged employing Matlab (MathWorks, Natick, MA) macros from the cSAXS beamline (http://www.psi.ch/sls/csaxs/software) to provide intensity versus momentum transfer q, defined as q = 4π/λ sin(θ/2), where θ is the scattering angle and λ is the wavelength of the incident X-rays. Afterward, background scattering was substracted using a buffer reference. The pair distance distribution function P(r) was calculated by GNOM.22 Three-dimensional low-resolution reconstructions were obtained using the ab initio bead modeling program GASBOR,23 which has been shown to be able to reliably generate shapes from SAXS data that are consistent with crystallographic X-ray structures.24 In our system, the theoretical scattering pattern of the models agreed well with the experimental scattering curves (χ2 =1.5 to 3.4). From multiple independent runs of GASBOR, similar widely branched structures like the ones depicted in Figure 4 were obtained.

ring of Phe residues. It varied in intensity and position during gelation. Time-resolved second-derivative absorption spectroscopy26 revealed a peak shift of ∼0.2 nm during the gelation process (Figure 1B). This shift indicated that upon Nsp1 hydrogel formation, Phe residues became less exposed to the solvent, that is, that the hydrophobicity around Phe residues increased over time.27 For comparison, a peak shift of about −0.4 nm is typically observed upon complete denaturation of a folded protein in Guanidinium-HCl.19 Normalized fluorescence emission spectra of the Nsp1 FG repeat domain (Nsp12−601) before and after hydrogel formation revealed three characteristic fluorescence emission maxima at 274, 282, and 290 nm, respectively (Figure 1C,D). They could be assigned to electronic transitions of the Phe residues from the singlet electronic excited state to the ground state.28 A small but significant red shift of these main emission bands by 2 nm indicated an increase in hydrophobicity around the Phe residues. A similar red shift is apparent when comparing an aqueous Phe solution with Phe present in the core of a folded protein.29 Remarkably, as compared with the liquid state, an additional, weak and structure-less band appeared in the gel state around 350 nm (Figure 1C, inset), which is typical for excimer emission.30−32 This strongly indicates that during gelation a vast part of the aromatic groups adopted an orientation appropriate for π−π stacking (excimer) interactions. This further suggests that also in the electronic ground state the Phe residues are in close proximity, allowing for their direct interaction. Most probably, they adopt parallel displaced or T-shaped geometries with typical distances ranging from 0.35 to 0.5 nm33 (Figure S1 in the Supporting Information). In conclusion, the results obtained from UV−VIS absorption and emission spectroscopy provided evidence of solvent dielectric alternations of the Phe microenvironment brought about by an increased shielding from the solvent during gelation. Hydrogelation Involves a Significant Transformation of Nonstructured Elements to β-Sheets. CD and infrared spectroscopy allow determining the secondary structure content within polypeptides. CD spectra recorded during Nsp1 hydrogel formation initially showed a low ellipticity in the region around 230 nm and an substantial minimum near 200 nm (Figure 2A), features typical for unfolded proteins.34 In contrast, the mature gel showed an evident contribution of βsheet domains. The small negative peak near 217 nm and the positive peak near 195 nm are associated with the (n, π*) and (π, π*) transitions in β-sheets, respectively.35 The data obtained before gelation are in accordance with earlier studies by Denning et al.36 However, possibly due to the drastically lower concentrations analyzed in their studies, these authors did not observe hydrogel formation and could thus not analyze the characteristics of the mature hydrogel. Quantitative analysis of our far-UV-CD data using the CDSSTR program package20 revealed an increase in the β-sheet content from ∼9 to ∼25% (Figure 2B, Table S2 in the Supporting Information) at the expense of unstructured contributions such as random coils and turns. Complementary structural information was obtained by timeresolved Fourier transform infrared spectroscopy (TR-FTIR), which is sensitive to coupled and uncoupled stretching and bending modes of amide bonds (Figure 2C). For an estimation of secondary structure contents within the ensemble, the amide I band (ν = 1700−1600 cm−1) was separated into its constituents. The spectrum recorded in D2O revealed an

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RESULTS Hydrogelation Involves π Stacking of Phe Residues in an Increasingly Hydrophobic Microenvironment. Given that Phe residues are essential for hydrogelation of the FG repeat domain from Nsp1p,14 it appeared to be reasonable to assume that their local microenvironment changes during the process. Optical UV−VIS absorption and fluorescence emission spectroscopy are sensitive methods to monitor such structural changes. Using these methods, we therefore expected to gain information about the local hydrophobicity change around Phe residues and potential aromatic interactions. In the UV absorption spectrum of the Nsp12−601 (Figure 1A), the dominating absorption band around 258 nm resulted from electronic transitions (1A1g→ 1B2u)25 within the benzene

Figure 1. Phe residues experience a more hydrophobic environment upon hydrogel formation of Nsp12−601. (A) UV−VIS absorption spectra of the liquid (black) and gel state (red) of Nsp12−601, respectively. The characteristic vibrational spectra of Phe are clearly resolved. (B) Time-resolved second-derivative absorption spectroscopy reveals a peak shift of the 258 nm Phe absorption band during gelation of Nsp12−601. This shift is diagnostic for a transfer of Phe residues into a more hydrophobic environment. Red line: fit according to monoexponential kinetics. (C) Normalized UV fluorescence spectra of the liquid (black) and gel state (red) of Nsp12−601, respectively. The inset showing the difference spectrum between both states suggests excimer formation between Phe residues. (D) Comparison between UV fluorescence spectra of the gel state of Nsp12−601 at 4 K and of an aqueous solution of a Phe-Gly dipeptide, showing the contribution of FG entities to the Nsp12−601 spectrum. For clarity, the spectra were normalized to the absorption at 281 nm. 1884

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not be unambiguously resolved by WAXS, possibly because of the small proportion of Phe residues (9%) within Nsp12−601. Nsp12−601 gel formation in vitro was also followed by timeresolved SAXS, which is sensitive to the morphology of the hydrogel network. Gelation was accompanied by the appearance of a peak in the low-q regime (Figure 3B), which signifies that this self-assembly process creates structures with a low periodic order. This feature indicates the existence of protein-poor and protein-rich microphases. The term “repetitive distance” will be used to denote the typical distance between these microphases and “correlation length” to describe their size. The pair distance distribution function P(r), an histogram of the pairwise distances44 of the branched protein assemblies, has been evaluated from the SAXS data of Nsp12−601 at various time points (Figure 3C). In this function, the shift of the peak maximum indicates an overall growth of the network. Concomitantly, the area under the curve, which is directly proportional to the volume of the gel phase, increases by a factor of 4.5. The oligomerization state of proteins in solution can also be monitored by the forward scattering value, that is, the extrapolated I(q = 0). This is an appropriate parameter because it is proportional to the average mass of the protein assemblies in solution. It showed a time-dependent increase in the apparent molecular mass, reflecting an increase in size of the assemblies (Figure 3D) during hydrogelation. A useful representation of the scattering pattern that serves as an indicator for the compactness of the solvated proteins44 is the Kratky plot (I (q)·q2 over q). It monitors the existence of globular or random coil-like conformers, respectively. The Kratky plot of Nsp12−601 before gelation lacked characteristic features, thereby indicating a high flexibility of the protein backbone (Figure 3E). These findings agree well with the classification of Nsp12−601 as an intrinsically disordered protein.36 During gelation two features emerged within the Kratky plot. The peak at q = 0.4 nm−1 was caused by the formation of smaller, partially folded entities, whereas the very intense maximum at q = 0.2 nm−1 could be linked to large oligomers possibly interconnected by flexible chains.45,46 The apparent shift of the peak at q = 0.2 nm−1 toward higher q values is a consequence of the superposition of the peak at higher q values originating from the self-assembly process of smaller entities which is described also by the P(r) function. In the gel state, coil-like elements were preserved and coexisted with an ensemble of oligomeric structures adopting molten globular shapes. To correlate these findings to a recent report predicting a collapsed-coil conformation for the N-terminal FG domains (Nsp11−172) and an extended-coil conformation within the highly charged FxFG (Nsp173−601) domain,47 hydrogels of similar Nsp1 subdomains were prepared and analyzed by SAXS. The Kratky plot of Nsp12−175 hydrogel displayed a shape typical for molten globular conformations (Figure 3F). In comparison, gels prepared from Nsp176−602 scattered weakly and showed a plateau typical for unstructured proteins with a high degree of random coils (Figure 3F). Both observations were therefore in line with the prediction.47 Importantly, the data demonstrated that the scattering pattern of Nsp2−601 is not simply the sum of the individual contributions but instead results from the complex interplay between both domains. The sharp maximum at low q values of the Nsp12−601 scattering pattern reflects a high degree of order within the assemblies, which might be induced by the electrostatic interactions within the highly

Figure 2. The β-sheet content of Nsp12−601 increases upon hydrogel formation. (A) Time-resolved far-UV CD measurements during hydrogel formation of Nsp12−601 showing a transformation from highly disordered conformers in the liquid state to more folded conformers with a significant β-sheet contribution. (B) The secondary structure content during the sol-gel transition of Nsp12−601 displayed as a function of time. A significant increase in β-sheet content on the expense of turn and random coil conformations is evident. We note that the concentration in the CD experiment had to be limited to 30 mg/mL due to the high absorbance, which precludes a direct comparison of the kinetics obtained with other techniques. (C) Fourier transform infrared (FTIR) spectra of Nsp12−601 in D2O medium. Observed spectra (scatter), decomposed bands (gray lines), and overall fit (red line) are depicted for the liquid state of Nsp12−601. The blue surface is attributed to β-sheet structures. (D) Difference spectra between the liquid and gel states of Nsp12−601 in H2O medium. The intense shoulder (arrow) around 3250 cm−1 indicates strong hydrogen bonding interactions.

increase in absorbance between 1625 and 1635 cm−1 during hydrogel formation, which could be unambiguously assigned to β-sheet structures.21 Additional IR spectra of Nsp12−601 recorded in H2O during gelation showed a decrease in the amide band attributed to N−H stretching vibrations of the peptide bond (3200−3500 cm−1). An intense shoulder visible around 3250 cm−1 (Figure 2D) indicated an involvement of N−H groups in strong hydrogen bondings.37 Although random-coil and unstructured features prevailed also in the gel state (Table S3 in the Supporting Information), it appeared that β-sheet elements and H-bonds discernibly contribute to a stabilization of the hydrogel network. Thereby the FTIR data confirm the results obtained by CD spectroscopy (Figure 2B, Table S2 in the Supporting Information). The Nsp1 Hydrogel Comprises Nanosized Pores and Nodes. Time-resolved X-ray scattering techniques38 allow probing the size and shape of macromolecules, ensembles, oligo- and polymers as well as their relative contribution in solution and noncrystalline materials.39,40 To analyze further the structural characteristics of the found β-sheet elements, we applied WAXS on the Nsp1 FG repeat domain during gelation. The scattering pattern of the Nsp12−601 hydrogel was characterized by two diffraction peaks at d = 1.34 nm and at d = 0.46 nm (Figure 3A), which corresponds to length scales typical for the peptide backbone separation between β-strands41 (0.4 to 0.5 nm) and between stacked βsheets42,43 (≈ 1.35 nm). Although clearly detected in fluorescence emission experiments (Figure 1C), (π, π) stacking interactions with characteristic distances of 0.35−0.5 nm could 1885

dx.doi.org/10.1021/bm300412q | Biomacromolecules 2012, 13, 1882−1889

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Figure 3. Long-range ordered structures evolve during Nsp1 hydrogel formation. (A) Wide-angle X-ray scattering (WAXS) pattern of the Nsp12−601 hydrogel. The peaks at d = 0.46 and 1.34 nm are attributed to the distances between the β-strands and between two stacked β-sheets, respectively. (B) Time-resolved small-angel X-ray scattering (SAXS) pattern of Nsp12−601 during gelation. The peak at q = 0.014 Å−1 indicates long-range ordered periodicity within the hydrogel. (C) Pair distribution functions P(r) obtained from the SAXS data. The length r refers to the pairwise distance of atoms within a particle. The maxima of the curves (symbols) shift to larger distances, thereby indicating an average growth of the protein assemblies. (D) The forward scattering I0 monitors the mass increase in ordered protein assemblies over time. (E) Kratky plots of the liquid to gel transition of Nsp12−601 at different time points. The very intense maximum (q = 0.014 Å−1) is attributed to specific large oligomers, whereas the weak shoulder (q ≈ 0.04 Å−1) represents smaller entities in a molten globular conformation. Unfolded monomers remain undetected. (F) The Kratky plot of Nsp1 repeat domains obtained at equal mass fractions. The plot of the N-terminal repeat domain (Nsp12−175) shows features typical for molten globular conformations. In contrast, Nsp1176−601 adopts a random-coil-like conformation, which is shown by the plateau at low intensity. Interplay of both domains is required to produce the scattering pattern of Nsp12−601.

reconstructions substantiated the idea that the Nsp1 FG repeat domain forms a 3-D protein meshwork with sieve-like elements. It is important to note that these models only visualize Nsp12−601 protomers present in spatially repetitive units (probably representing 60%) prevail in irregular and probably flexible structures, possibly imparting flexibility and accessibility to the gel. Separate analysis of the N-terminal FG-rich domain (Nsp12−175) and the FxFG domain (Nsp176−601) by Kratky plots showed a molten globular conformation only for the Nterminal domain. Therefore, it can be concluded that ordered structures comprising β-sheet elements are most likely formed from the Nsp12−175 domain. However, an interplay of the cohesive Nsp12−175 domain with the highly charged and unstructured domain (Nsp176−601) leads to the formation of long-range ordered repetitive structures (Figure 5). Given that

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CONCLUSIONS All nucleocytoplasmic transport processes within eukaryotic cells proceed through NPCs containing a permeability barrier that is constituted by long, nonglobular nucleoporin FG repeat domains. The isolated FG repeat domain of the yeast nucleoporin Nsp1p forms noncovalently cross-linked hydrogels that can reproduce the permeability properties of authentic NPCs. In this work, we have characterized the self-assembly and structural organization of such Nsp1-derived FG hydrogels. Combining time-resolved optical spectroscopy (time-resolved optical absorption, optical emission, and CD spectroscopy) as well as time-resolved X-ray scattering techniques, we found that the formation of such hydrogels involves structural rearrangements on all addressed length scales. The mature hydrogel is a composite material comprising protein-rich and protein-poor microphases. Our results suggest that the protein-rich microphase stabilizes the hydrogel and contains rigid, scaffold-like elements rich in β-like structures. The protein-poor phase, in contrast, seems to be dominated by less ordered protein chains that might provide flexibility and accessibility to the network. We discuss possible functional implications of this structural arrangement. We propose that the protein-poor microphase represents the preferred path for passage of inert macromolecules and that the pores formed within this phase are the main determinant for the observed sieve-like size selectivity of the Nsp1 hydrogel. We speculate that NTRs easily overcome the size limits set by this barrier because they interact with FG repeat domains and can therefore open meshes that are thermodynamically inaccessible for inert molecules. Depending on the strength of NTR•FG repeat interactions, NTRs might also be able to resolve the tight interactions dominating the protein-rich microphase.

Figure 5. The morphology of the hydrogel is determined by the balance of cohesive and repulsive domains. Nsp12−175 forms inhomogeneous gels with compact assemblies of various sizes (top left panel, orange) while Nsp1176−602 forms a random coil-like network (top right, blue). The interplay of electrostatic repulsion within Nsp1176−602 and hydrophobic attraction within Nsp12−175 (lower panel) creates a new morphology characterized by a long-range ordered structure of alternating protein-rich and protein-poor regions (bottom).

Nsp12−175 showed a strong tendency to flocculate out of solution at nonsaturating concentrations, we suggest that the charged and repulsive elements present in Nsp1176−601 prevent a collapse of the entire FG domain to tightly packed aggregates. In this study, we further presented evidence that the Nsp1 FG repeat hydrogel is a composite material comprising proteinrich, scaffold-like elements as well as a microphase with a lower local protein concentration. This “protein-poor” microphase appears to be dominated by protein domains lacking defined secondary structures and presumably forms a continuous system of channels within the hydrogel. How can macromolecules pass this composite material? In vitro, the Nsp1 FG repeat hydrogel behaves as a selective filter with permeability properties similar to authentic NPCs; that is, it excludes inert material ≥5 nm but allows high load of transport of even large NTR•cargo complexes.12 By definition, inert material does not physically interact with the hydrogel. As 1888

dx.doi.org/10.1021/bm300412q | Biomacromolecules 2012, 13, 1882−1889

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We assume that the blend of FG repeat domains within authentic NPCs will self-assemble into structures similar to those observed within the Nsp1 hydrogel. We thus expect that the proposed mechanisms explaining selective transport will also apply to the in vivo situation.

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ASSOCIATED CONTENT

* Supporting Information S

Figure visualizing possible geometries for Phe-Phe interactions; tables showing the secondary structural content and text explaining the evaluation of the average mesh size from the SAXS pattern are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +495512011268. Fax: +49 (551) 2011501. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the SFB755 Nanoscale Photonic Imaging of the DFG. We thank Jürgen Schünemann and Sissi Sonnenkalb for excellent technical assistance.

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REFERENCES

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dx.doi.org/10.1021/bm300412q | Biomacromolecules 2012, 13, 1882−1889