Summary

Highly selective nucleocytoplasmic molecular transport is critical to eukaryotic cells, which is illustrated by size-filtering diffusion and karyopherin-mediated passage mechanisms. However, a considerable number of large proteins without nuclear localization signals are localized to the nucleus. In this paper, we provide evidence for the spontaneous migration of large proteins in a karyopherin-independent manner. Time-lapse observation of a nuclear transport assay revealed that several large molecules spontaneously and independently pass through the nuclear pore complex (NPC). The amphiphilic motifs were sufficient to overcome the selectivity barrier of the NPC. Furthermore, the amphiphilic property of these proteins enables altered local conformation in hydrophobic solutions so that elevated surface hydrophobicity facilitates passage through the nuclear pore. The molecular dynamics simulation revealed the conformational change of the amphiphilic structure that exposes the hydrophobic amino acid residues to the outer surface in a hydrophobic solution. These results contribute to the understanding of nucleocytoplasmic molecular sorting and the nature of the permeability barrier.

Introduction

Nucleocytoplasmic communication is critical to both basal and adaptive activities of eukaryotic cells, which is accomplished by molecular transport through the nuclear pore complex (NPC). The NPC is a symmetric, octameric structure that is embedded in the nuclear envelope with an outer diameter of ∼120 nm and a height of ∼200 nm. The inside of the pore is composed of characteristic subunits that contain hydrophobic phenylalanine-glycine motifs (FG-Nups) and serves as a selective barrier for molecular trafficking (Denning et al., 2003). One of the well-known selective properties of the NPC is size dependent filtration. Water, ions and proteins smaller than ∼40 kDa can pass through the NPC via passive diffusion, whereas proteins larger than ∼40 kDa rarely diffuse through the NPC (Mohr et al., 2009). On the other hand, karyopherin-mediated nuclear transport pathways are known to strongly influence the subcellular distribution of proteins. Karyopherins recognize specific cargo, such as proteins containing nuclear localization signals (NLS, recognized by importin β) and nuclear export signals (NES, recognized by CRM1), and mediate the passage through the NPC via hydrophobic interactions with FG-Nups (Mosammaparast and Pemberton, 2004).

However, these canonical nuclear transport pathways are not sufficient to explain the general nuclear localization of proteins. Bioinformatic analysis revealed that among ≈1500 proteins with localization restricted to the nucleus in Saccharomyces cerevisiae, 57% contain a classical NLS with the remainder expected to use another mechanism to enter the nucleus (Lange et al., 2007). In addition to proteins constrained to the nucleus, there are also relevant examples of nuclear transport of cytoplasmic/cytoskeletal proteins. Interestingly, evidence for the association of large cytoskeletal proteins with nuclear functions has been accumulating (Kumeta et al., 2012). Actinin-4 interacts with INO80 chromatin remodeling complex and regulates gene expressions, whereas several different forms of spectrin are involved in DNA repair and the formation of nuclear bodies. Nuclear translocation of β-catenin, primarily a focal adhesion component, is a constituent of the canonical Wnt signaling pathway, which is an indispensable step for asymmetric cell division during embryonic development. Such protein classes often contain amphiphilic helices and include: i) cytoskeletal proteins containing spectrin repeats (Kumeta et al., 2010); ii) nuclear shuttling/signaling molecules containing armadillo repeats (Yokoya et al., 1999); and iii) a group of proteins containing HEAT repeats, to which, interestingly, the karyopherins themselves belong. Recently, it was revealed through the use of chemically modified BSA that molecular surface hydrophobicity is sufficient to overcome the selectivity barrier of the NPC (Naim et al., 2009). In this study, we focused on the amphiphilic nature of certain proteins and found that they undergo a conformational change to adapt to a hydrophobic environment, with a resulting increase in surface hydrophobicity facilitating spontaneous passage through the NPC.

Results and Discussion

Karyopherin-independent nuclear transport of large proteins

A nuclear transport assay has been developed for analyzing nuclear transport of proteins using digitonin-treated semi-permeabilized cells (Adam et al., 1990). We utilized this assay in time-lapse observations to quantitatively analyze the nuclear transport of large molecules and transport kinetics. Full-length cDNAs for actinin-4, βI-spectrin, and β-catenin were cloned and GFP-fused recombinant proteins were purified using the Sf9 insect cell expression system (Fig. 1A). Time-lapse observation of the nuclear import of purified proteins clearly showed that these proteins continuously migrate into the nucleus in a karyopherin-independent manner (Fig. 1B,C). GFP-importin-β rapidly accumulated in the nucleus in a few minutes. It showed slightly stronger signals at the nuclear envelope, due to its docking activity to the pore (Nachury and Weis, 1999). Compared to GFP-importin-β, actinin-4, βI-spectrin, and β-catenin showed slower nuclear transport. It should be noted that these proteins exceed the size limitation for passive diffusion through the NPC, and the assay solution does not contain any transport mediators. Fibrous signals of GFP-actinin-4 outside the nucleus at 300 and 700 seconds are derived from its binding to actin cytoskeleton. β-Catenin showed aggregated fluorescent signals during the incubation, probably due to its self-association property under this experimental condition, or some remaining binding partners in the semi-permeabilized cells. GFP was used as a passively diffusing control. Fluorescein-conjugated 70 kDa dextran, a hydrophilic polysaccharide, serves as a negative control that is excluded from the nucleus. Analyses of nuclear efflux, observed after removing the extracellular fluorescent molecules, showed that the signals from actinin-4, βI-spectrin, and β-catenin were decreased (Fig. 1D). These results demonstrated that nuclear shuttling of these molecules, both import and export, can be achieved in a karyopherin-independent manner. Importin-β was not actively exported from the nucleus under this experimental condition, which may be due to strong binding to nuclear factors or the depletion of RanGTP in the nucleus. The signal intensities were quantified and rate constants were calculated by curve fitting. Rate constants for import (kin) for actinin-4, βI-spectrin, and β-catenin were 5.27, 5.06 and 4.69 (10−3 s−1), approximately six times less than that of importin-β (Fig. 1E, 31.86). Rate constants for export (kout) were estimated by analyzing signal influx and efflux, and were similar for all proteins with the exception of importin-β. Namely, several large molecules spontaneously and independently passed through the NPC. Compared to the GFP control that showed similar rates of import and export, the values for other proteins used in this experiment were significantly different, suggesting that their passages are not the simple diffusion, and their imports and exports are regulated in different manners.

Fig. 1.

Nuclear entry of large non-nuclear localization signal proteins. (A) GFP and GFP-fused actinin-4, βI-spectrin and β-catenin are expressed in the Sf9 insect cell expression system and purified. Proteins were loaded onto 5% to 20% acrylamide gels and visualized with Coomassie brilliant blue. (B) Time-lapse observations of nuclear transport of GFP-fused proteins in semi-permeabilized HeLa cells. The images were acquired for approximately 12 minutes. Images show before and after adding the proteins (Scale bar: 10 µm). Fluorescein-conjugated 70 kDa dextran is the negative control. (C) The averaged nuclear fluorescent signal. Signal intensities of ten different cells per experiment were measured in three independent experiments. Error bar represents the standard deviation (s.d.) (D) Efflux of nuclear GFP fluorescence, analyzed by removing the fluorescent molecules after the import assay. The data were processed as described for (C). (E) The rate constants for import (kin) and export (kout) are calculated by curve fitting the influx and efflux data, as described in the Materials and Methods section.

Fig. 1.

Nuclear entry of large non-nuclear localization signal proteins. (A) GFP and GFP-fused actinin-4, βI-spectrin and β-catenin are expressed in the Sf9 insect cell expression system and purified. Proteins were loaded onto 5% to 20% acrylamide gels and visualized with Coomassie brilliant blue. (B) Time-lapse observations of nuclear transport of GFP-fused proteins in semi-permeabilized HeLa cells. The images were acquired for approximately 12 minutes. Images show before and after adding the proteins (Scale bar: 10 µm). Fluorescein-conjugated 70 kDa dextran is the negative control. (C) The averaged nuclear fluorescent signal. Signal intensities of ten different cells per experiment were measured in three independent experiments. Error bar represents the standard deviation (s.d.) (D) Efflux of nuclear GFP fluorescence, analyzed by removing the fluorescent molecules after the import assay. The data were processed as described for (C). (E) The rate constants for import (kin) and export (kout) are calculated by curve fitting the influx and efflux data, as described in the Materials and Methods section.

The amphiphilic spectrin repeat is sufficient for nuclear targeting

A feature that is common to these proteins is their amphiphilic property, which is due to the presence of amphiphilic α-helices. β-Catenin contains armadillo repeats and does not require karyopherins for the nuclear targeting (Malik et al., 1997; Yokoya et al., 1999). Actinins and spectrins possess multiple spectrin repeats (SRs), which consist of three amphiphilic α-helices bundled in a rod-shaped structure through the interaction of each hydrophobic surface of the helices (Yan et al., 1993).

To test if the amphiphilic motif itself is generally permeable to the NPC, ten different SR regions from four typical SR proteins (actinin-1, actinin-4, βI-spectrin and dystrophin) were chosen to assess the role of amphiphilic SR regions in nuclear targeting (Fig. 2A). Expression of GFP-fused fragments containing three to six SRs in HeLa cells resulted in consistent nuclear localization (Fig. 2B). In comparison to results obtained using a protein containing three tandem GFP repeats that lacked an NLS and was not found in the nucleus, it appears that SRs are capable of mediating nuclear targeting.

Fig. 2.

Nuclear transport of amphiphilic spectrin repeat fragments. (A) Ten multiple spectrin repeat (SR)-containing regions from actinin-1, actinin-4, βI-spectrin and dystrophin (indicated by Roman numbers “i” through “x”) were subcloned into GFP-fusion expression vectors. The fragments contain the following amino acid regions from their respective proteins: (i) 286–751; (ii) 267–732; (iii) 302–953; (iv) 955–1590; (v) 1592–2129; (vi) 342–666; (vii) 722–1262; (viii) 1269–1779; (ix) 1784–2428; and (x) 2473–3039. SRs are indicated by boxes and are numbered. (B) The multiple SR-containing fragments in (A) are expressed in HeLa cells. GFP and a protein containing three tandem GFP repeats are expressed in HeLa cells, with and without fusion of a nuclear localization signal (NLS) as controls. Scale bars: 10 µm. (C) Nuclear transport of multiple SR-containing fragments (as indicated in A) purified from a bacterial expression system. The semi-permeabilized cells were washed and fixed, after incubating them with the fragments. Scale bars: 10 µm. (D) Nuclear transport assay in the presence of an importin-β mutant (IBM). The IBM was pre-incubated prior to the incubation with the fragments. (Each scale bar is 10 µm.) (E) Quantitation of the effect of an IBM on the nuclear transport of SR fragments. Error bar represents the standard deviation (s.d.) (F) A concentration-dependent effect of IBM on nuclear transport of GFP-βI spectrin SR1-6. (G)kin values in the presence of IBM are calculated by curve fitting, as described in the Materials and Methods section.

Fig. 2.

Nuclear transport of amphiphilic spectrin repeat fragments. (A) Ten multiple spectrin repeat (SR)-containing regions from actinin-1, actinin-4, βI-spectrin and dystrophin (indicated by Roman numbers “i” through “x”) were subcloned into GFP-fusion expression vectors. The fragments contain the following amino acid regions from their respective proteins: (i) 286–751; (ii) 267–732; (iii) 302–953; (iv) 955–1590; (v) 1592–2129; (vi) 342–666; (vii) 722–1262; (viii) 1269–1779; (ix) 1784–2428; and (x) 2473–3039. SRs are indicated by boxes and are numbered. (B) The multiple SR-containing fragments in (A) are expressed in HeLa cells. GFP and a protein containing three tandem GFP repeats are expressed in HeLa cells, with and without fusion of a nuclear localization signal (NLS) as controls. Scale bars: 10 µm. (C) Nuclear transport of multiple SR-containing fragments (as indicated in A) purified from a bacterial expression system. The semi-permeabilized cells were washed and fixed, after incubating them with the fragments. Scale bars: 10 µm. (D) Nuclear transport assay in the presence of an importin-β mutant (IBM). The IBM was pre-incubated prior to the incubation with the fragments. (Each scale bar is 10 µm.) (E) Quantitation of the effect of an IBM on the nuclear transport of SR fragments. Error bar represents the standard deviation (s.d.) (F) A concentration-dependent effect of IBM on nuclear transport of GFP-βI spectrin SR1-6. (G)kin values in the presence of IBM are calculated by curve fitting, as described in the Materials and Methods section.

The GFP-fused SR fragments were also assessed with a semi-intact nuclear transport assay. After incubation with GFP-fragments, semi-intact cells were washed and fixed before observation. All fragments accumulated in the nucleus, demonstrating the capability for spontaneous nuclear transport despite exceeding the theoretical size limitation of the NPC (Fig. 2C). A dominant-negative importin-β mutant (IBM), which lacks cargo-binding and Ran-binding domains, is known to inhibit karyopherin-dependent nuclear transport by masking hydrophobic subunits of the NPC (Kutay et al., 1997). When IBM was added to the assay, nuclear GFP signal was significantly decreased, (Fig. 2D,E, ≈50% on average). IBM-mediated suppression was found to be concentration dependent (Fig. 2F), with the kin values for GFP-βI-spectrin SR1-6 decreasing with increasing IBM concentrations (Fig. 2G).

The semi-intact nuclear transport assay clearly demonstrated the spontaneous nuclear targeting of amphiphilic SR fragments (Fig. 2C). Some of these fragments tend to accumulate in the nucleus when they are expressed in HeLa cells (Fig. 2B), while the full length SR proteins are equilibrated in the transport assay (Fig. 1B). In live cells, unidentified transport mechanisms may contribute to such accumulation, in addition to their spontaneous passage. Another possibility is that such fragments are retained in the nucleus by interacting with binding partners, which are easily removed from the nucleus during digitonin-permeabilization in the semi-intact assay.

Amphiphilic proteins increase their surface hydrophobicity in hydrophobic solutions

A hydrophobic fluorescent probe, 4,4′-bis-1-anilinonaphthalene 8-sulfonate (bis-ANS), is known to emit fluorescence when it binds to a hydrophobic surface on a molecule. When equal amounts of purified proteins were subjected to bis-ANS measurement, GFP-fused actinin-4, βI-spectrin, and β-catenin showed higher fluorescent signal intensities compared to GFP alone or GFP-GST, consistent with higher surface hydrophobicity (Fig. 3A). When the bis-ANS measurements were performed in a hydrophobic environment in the presence of increasing amounts of trifluoroethanol (TFE), the fluorescence from GFP and GFP-GST were decreased due to attenuation of hydrophobic interactions between the probe and proteins. Interestingly, importin-β and β-catenin exhibited increased bis-ANS binding in the presence of 5% to 10% TFE, and actinin-4 exhibited slightly enhanced fluorescent signal in 5% to 15% TFE compared to controls. Relative signal intensities compared with GFP alone clearly demonstrated increased surface hydrophobicity of amphiphilic proteins in a hydrophobic environment (Fig. 3B).

Fig. 3.

Conformational changes of amphiphilic proteins in a hydrophobic solution. (A) Surface hydrophobicity of amphiphilic proteins, as evaluated by measuring the fluorescence signals of bis-ANS incubated with 2 µg of each protein in PBS supplemented with TFE (0% to 50%, v/v). Measurements were repeated three times and values are presented as the mean ± standard deviation (s.d.). (B) Relative values of the bis-ANS signal intensities against GFP in the presence of 0%, 5% and 10% TFE. Values of three independent experiments are represented as the mean ± s.d. (C) Helical wheel model of the third spectrin repeat of actinin-4, based on a NMR model (PDB: 1WLX). Hydrophobic amino acids are indicated in gray and hydrophilic amino acids in blue. (D) Molecular structure of the third SR of actinin-4 in a high permittivity condition (e = 70), as revealed by a molecular dynamics simulation. The entire structure is represented in a ribbon model (left), and the zoomed image is shown with hydrophobic amino acid residues indicated in red (right). The three helices are indicated as H1, H2, and H3. The indicated amino acids are A21, F24, W27, M28, A31, M32, L35, M38-V41, I44, I47, L50, I51, A53, F57, A64, W107, V110, and V114. (E) The molecular structure of the third SR of actinin-4 in a low permittivity condition (e = 20) is shown in the same manner as in (D). (F) The circular dichroism (CD) spectra of actinin-4 and βI-spectrin in PBS (lined in blue) or PBS supplemented with 50% v/v TFE (red). The molar ellipticity (deg cm2 dmol−1) at 222 nm were −38,662 and −40,776 (actinin-4; in PBS and PBS+50% TFE, respectively), and −36,147 and −36,175 (βI-spectrin, in PBS and PBS+50% TFE, respectively).

Fig. 3.

Conformational changes of amphiphilic proteins in a hydrophobic solution. (A) Surface hydrophobicity of amphiphilic proteins, as evaluated by measuring the fluorescence signals of bis-ANS incubated with 2 µg of each protein in PBS supplemented with TFE (0% to 50%, v/v). Measurements were repeated three times and values are presented as the mean ± standard deviation (s.d.). (B) Relative values of the bis-ANS signal intensities against GFP in the presence of 0%, 5% and 10% TFE. Values of three independent experiments are represented as the mean ± s.d. (C) Helical wheel model of the third spectrin repeat of actinin-4, based on a NMR model (PDB: 1WLX). Hydrophobic amino acids are indicated in gray and hydrophilic amino acids in blue. (D) Molecular structure of the third SR of actinin-4 in a high permittivity condition (e = 70), as revealed by a molecular dynamics simulation. The entire structure is represented in a ribbon model (left), and the zoomed image is shown with hydrophobic amino acid residues indicated in red (right). The three helices are indicated as H1, H2, and H3. The indicated amino acids are A21, F24, W27, M28, A31, M32, L35, M38-V41, I44, I47, L50, I51, A53, F57, A64, W107, V110, and V114. (E) The molecular structure of the third SR of actinin-4 in a low permittivity condition (e = 20) is shown in the same manner as in (D). (F) The circular dichroism (CD) spectra of actinin-4 and βI-spectrin in PBS (lined in blue) or PBS supplemented with 50% v/v TFE (red). The molar ellipticity (deg cm2 dmol−1) at 222 nm were −38,662 and −40,776 (actinin-4; in PBS and PBS+50% TFE, respectively), and −36,147 and −36,175 (βI-spectrin, in PBS and PBS+50% TFE, respectively).

A helical wheel model of actinin-4 SR3, based on a known NMR structure (PDB: 1WLX), showed significantly higher hydrophobic amino acid content in the inner face of the helical bundle compared to the outer surface (Fig. 3C). Starting with this NMR structure, molecular dynamics simulations were performed to survey conformational changes in SR. In the simulations, the permittivity constants (represented as “e”) of the solution were set to 70 and 20. In a hydrophilic solution (e = 70), the three helices are closely aligned and hydrophobic amino acid residues are folded inside the bundle (Fig. 3D, hydrophobic amino acid residues are shown in red). In contrast, in hydrophobic environments (e = 20), the helices are bent and exhibit an open structure that exposes the hydrophobic residues to the outer surface (Fig. 3E). This simulation analysis supports our experimental results concerning the surface properties of amphiphilic proteins.

Ultraviolet circular dichroism (CD) spectroscopy was performed to analyze the helical contents of proteins in hydrophobic environment. The negative peak of CD angle at 222 nm is known to represent the helical contents (Greenfield, 2006). Both actinin-4 and βI-spectrin did not show significant differences of this value in 0% and 50% TFE-containing solutions (−38,662 to −40,776, and −36,147 to −36,175, respectively) (Fig. 3F), suggesting that each helix was not disrupted in this experimental condition. The helical contents in each solution were estimated to be 44.2% and 48.1%, and 45.4% and 47.1%, for actinin-4 and βI-spectrin, respectively, based on the spectrum analyses using K2D3 prediction program (Louis-Jeune et al., 2011). Combined with surface hydrophobicity measurement and molecular dynamics simulation, it is reasonable to conclude that configurational change in amphiphilic helices is sufficient to increase surface hydrophobicity of the protein to facilitate spontaneous transport through the NPC. We propose that such dynamic conformational change in the molecules is an important factor for NPC permeability, including the transport of karyopherins themselves.

Biological implication of spontaneous molecular migration into the nucleus

There is increasing evidence showing the relationship between molecular surface hydrophobicity and NPC permeability. Karyopherins are known to have a similar molecular conformation and a greater surface hydrophobicity than other cytoplasmic proteins (Ribbeck and Görlich, 2002). Hydrophobic interactions are expected to be a major driving force for the passage of importin-β through the NPC, given that it interacts with hydrophobic nucleoporins (Bayliss et al., 2000; Bayliss et al., 2002; Bednenko et al., 2003; Otsuka et al., 2008). These findings suggest the possibility that any protein that interacts hydrophobically with the NPC can pass through the pore unaided. Indeed, BSA chemically modified to increase its surface hydrophobicity was shown to overcome the selectivity barrier of the NPC (Naim et al., 2009). Considering that hydrophobic interaction is one of the major driving forces for protein folding/self-assembly in general (Gerstman and Chapagain, 2005), it is likely that a variety of proteins capable of undergoing conformational changes to adapt to the hydrophobic environment of the NPC can spontaneously migrate in and out of the nucleus.

In contrast to the Brownian ratchet mechanism of molecular transport into the mitochondria, it is believed that molecules maintain their conformation during passage through the NPC. However, we propose that certain molecules alter their surface hydrophobicity by undergoing a local conformational change, enabling spontaneous migration into the nucleus (Fig. 4A,B). This mode of passage through the NPC is likely to be involved in many important cellular events. Nuclear export-based mechanisms seem to play a dominant role in subcellular localization of several nuclear shuttling proteins (Nix and Beckerle, 1997; Petit et al., 2005; Stüven et al., 2003; Wada et al., 1998). We postulate that the nuclear border is more permissive than previously thought, and that many proteins are actually capable of migrating into the nucleus. This spontaneous migration model will provide important clues for unveiling the nature of the NPC, as well as understanding complicated molecular sorting mechanisms in cells, which are carefully orchestrated to achieve basal and adaptive cellular functions.

Fig. 4.

The spontaneous migration model. (A) Spontaneous migration is a carrier-free transport system for proteins with amphiphilic properties. These molecules increase their surface hydrophobicity in hydrophobic environments, which provide enhanced interaction with FG-Nups. (B) Properties of transport pathways through the nuclear pore complex. While karyopherin itself spontaneously passes through the pore by increasing its surface hydrophobicity, it is unclear whether its cargo also adapts to the hydrophobic environment. Spontaneous migration is a bidirectional passage mechanism. Actual localization of the protein in intact cells may be determined by release/retention in the nucleoplasm or cytoplasm, in addition to its spontaneous migrating property.

Fig. 4.

The spontaneous migration model. (A) Spontaneous migration is a carrier-free transport system for proteins with amphiphilic properties. These molecules increase their surface hydrophobicity in hydrophobic environments, which provide enhanced interaction with FG-Nups. (B) Properties of transport pathways through the nuclear pore complex. While karyopherin itself spontaneously passes through the pore by increasing its surface hydrophobicity, it is unclear whether its cargo also adapts to the hydrophobic environment. Spontaneous migration is a bidirectional passage mechanism. Actual localization of the protein in intact cells may be determined by release/retention in the nucleoplasm or cytoplasm, in addition to its spontaneous migrating property.

Materials and Methods

cDNA constructs and recombinant proteins

Complementary DNA for GFP was digested from pEGFP-C1 vector at NcoI/BglII sites and cloned into NcoI/BamHI sites of pFastBac-HT-B vector. cDNAs for actinin-4, βI-spectrin, and β-catenin were amplified by PCR from HeLa total RNA reverse transcribed with the SuperScript First-Strand Synthesis System (Invitrogen). cDNA for dystrophin was purchased from Kazusa DNA Research Institute. The cDNAs were cloned into the pEGFP-C1 vector for expression in HeLa cells. For protein purification, cDNAs were cloned into the pFastBac-HT-B-GFP vector and expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system (Invitrogen).

Nuclear transport assay

Semi-permeabilized cells were prepared by digitonin treatment, as previously described (Kumeta et al., 2010). 70 kDa fluorescein- and rhodamine B-conjugated dextran were purchased from Molecular Probes. Microscopic observations were performed at room temperature using a confocal laser-scanning microscope (LSM 5 PASCAL, Carl Zeiss) with a 63×Plan-Apo objective. Time-lapse images were taken after incubation with transport substrates. Signal intensities of ten different nuclei were quantified and statistically analyzed. Curve fitting was performed to obtain rate constants for import (kin) and export (kout) by applying the following formula: Y = exp(−tkout)Y0 + (kin/kout)×{1 - exp(−tkout)} where, X: cytoplasmic (background) signal intensity; Y: nucleoplasmic signal intensity; t: time; and Y0: Y at time = 0.

Surface hydrophobicity measurement

The hydrophobic fluorescent probe bis-ANS was purchased from Sigma. Proteins (2 µg) were dissolved in 50 µl PBS containing 0% to 50% v/v TFE and incubated for 5 minutes. Ten micromolar bis-ANS was added and incubated for 5 minutes. Fluorescence was quantified using a plate reader (Wallac 1420 ARVO SX, Perkin Elmer) with excitation and emission wavelengths of 405 and 460 nm, respectively.

Molecular dynamics simulation

The NMR structure of SR was obtained from a protein structure database (PDB: 1WLX). Model 1 of 20 was selected for analysis. The simulation was performed using the Amber11 molecular dynamics package and AmberTools (version 1.5) (Case et al., 2010). Two Na+ ions were placed around SR as counter ions. After short energy minimizations, three production runs were conducted using the generalized Born model (Hawkins et al., 1995; Hawkins et al., 1996; Tsui and Case, 2001) with dielectric constants of 70 and 20. The time increment was 1 fs and trajectories were calculated up to 10 ns. No cut-off for long-range interactions was set. The temperature was kept at 300K using the Langevin thermostat (collision frequency = 1.0 ps−1).

CD spectroscopy

cDNA for actinin-4 was cloned into pGEX-5X vector (GE) and expressed in bacteria. After purification by glutathione sepharose beads (GE), GST-tag was excised with Factor Xa protease (GE). βI-Spectrin was cloned into pFastBac-HT-B vector, expressed in Sf9 cells, and purified with Ni-NTA agarose (Quiagen). CD spectra were obtained with a J-805 Spectropolarimeter (JASCO) with temperature controller using a 1 mm optical cuvette. Spectra were recorded from 240 nm to 200 nm at 25°C, using 0.05 mg/ml purified proteins in PBS or PBS with 50% v/v TFE. An average of five runs was obtained by sampling every 0.2 nm with 10 nm/min scanning speed.

Acknowledgements

We thank Dr Shotaro Otsuka and Mr Yutaka Takashima for helpful discussions and technical support.

Funding

This work was supported by a Grant-in-Aid for Research Activity Start-up (to M.K.) from The Japan Society for the Promotion of Science (JSPS); a Grant-in-Aid for Scientific Research on Innovative Areas “Spying minority in biological phenomena” [grant number 3306: 24115512 to M.K.] from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; a Grant-in-Aid for Scientific Research on Innovative Areas “Virus-Host Cell Competency” [grant number 3405: 24115003 to K.T.] from MEXT, Japan; and the Funding Program for Next Generation World-Leading Researchers (to S.H.Y.) of MEXT, Japan.

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