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• W2068918178 abstract "Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1–4). The amino acid substitution A546T in the FAS1–4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1–4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1–4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation. Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1–4). The amino acid substitution A546T in the FAS1–4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1–4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1–4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation. Transforming growth factor β induced protein (TGFBIp) 2The abbreviations used are: TGFBIptransforming growth factor β-induced proteinFAS1–4fourth fasciclin 1 domain of TGFBIpThTThioflavin TANS1-anilinonapthalene-8-sulfonateAF4asymmetrical flow field-flow fractionationDOPC1,2-dioleoyl-sn-glycero-3-phosphocholineDOPG1,2-dioleoyl-sn-[phosphor-rac-(1-glycerol)]TEMtransmission electron microscopy. is an extracellular protein of 72 kDa (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. cDNA cloning and sequence analysis of β ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-β.DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (501) Google Scholar) expressed in the cornea with no post-translational modifications (2Escribano J. Hernando N. Ghosh S. Crabb J. Coca-Prados M. cDNA from human ocular ciliary epithelium homologous to β ig-h3 is preferentially expressed as an extracellular protein in the corneal epithelium.J. Cell. Physiol. 1994; 160: 511-521Crossref PubMed Scopus (152) Google Scholar, 3Andersen R.B. Karring H. Møller-Pedersen T. Valnickova Z. Thøgersen I.B. Hedegaard C.J. Kristensen T. Klintworth G.K. Enghild J.J. Purification and structural characterization of transforming growth factor β induced protein (TGFBIp) from porcine and human corneas.Biochemistry. 2004; 43: 16374-16384Crossref PubMed Scopus (40) Google Scholar). It is purified from mammalian cells as a monomer although it can form higher order structures such as dimers and trimers in vitro (4Basaiawmoit R.V. Oliveira C.L. Runager K. Sørensen C.S. Behrens M.A. Jonsson B.H. Kristensen T. Klintworth G.K. Enghild J.J. Pedersen J.S. Otzen D.E. SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the R124H mutation.J. Mol. Biol. 2011; 408: 503-513Crossref PubMed Scopus (19) Google Scholar). Numerous links have been made between corneal dystrophies and TGFBIp through protein and genetic analyses (5Klintworth G.K. Valnickova Z. Enghild J.J. Accumulation of β ig-h3 gene product in corneas with granular dystrophy.Am. J. Pathol. 1998; 152: 743-748PubMed Google Scholar, 6Munier F.L. Korvatska E. Djemaï A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies.Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (519) Google Scholar, 7Kannabiran C. Klintworth G.K. TGFBI gene mutations in corneal dystrophies.Hum. Mutat. 2006; 27: 615-625Crossref PubMed Scopus (125) Google Scholar, 8Klintworth G.K. The molecular genetics of the corneal dystrophies. Current status.Front. Biosci. 2003; 8: d687-d713Crossref PubMed Scopus (145) Google Scholar). TGFBIp has an N-terminal cysteine-rich region, four consecutive fasciclin-1 (FAS1) domains, and an integrin-binding motif, RGD, near the C terminus (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. cDNA cloning and sequence analysis of β ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-β.DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (501) Google Scholar). The four FAS1 domains are homologous to each other and to fasciclin-1 from Drosophila (9Zinn K. McAllister L. Goodman C.S. Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila.Cell. 1988; 53: 577-587Abstract Full Text PDF PubMed Scopus (302) Google Scholar), whose crystal structure contains seven β-strands and five α-helices (10Clout N.J. Tisi D. Hohenester E. Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I.Structure. 2003; 11: 197-203Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). transforming growth factor β-induced protein fourth fasciclin 1 domain of TGFBIp Thioflavin T 1-anilinonapthalene-8-sulfonate asymmetrical flow field-flow fractionation 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-dioleoyl-sn-[phosphor-rac-(1-glycerol)] transmission electron microscopy. TGFBIp-associated dystrophies affect various layers of the cornea, causing visual impairment (11Weiss J.S. Møller H.U. Lisch W. Kinoshita S. Aldave A.J. Belin M.W. Kivelä T. Busin M. Munier F.L. Seitz B. Sutphin J. Bredrup C. Mannis M.J. Rapuano C.J. Van Rij G. Kim E.K. Klintworth G.K. The IC3D classification of the corneal dystrophies.Cornea. 2008; 27: S1-S83Crossref PubMed Scopus (268) Google Scholar, 12Conrad T.J. Chandler D.B. Corless J.M. Klintworth G.K. In vivo measurement of corneal angiogenesis with video data acquisition and computerized image analysis.Lab. Invest. 1994; 70: 426-434PubMed Google Scholar). TGFBIp-linked dystrophies are inherited in an autosomally dominant manner and are phenotypically heterogeneous (11Weiss J.S. Møller H.U. Lisch W. Kinoshita S. Aldave A.J. Belin M.W. Kivelä T. Busin M. Munier F.L. Seitz B. Sutphin J. Bredrup C. Mannis M.J. Rapuano C.J. Van Rij G. Kim E.K. Klintworth G.K. The IC3D classification of the corneal dystrophies.Cornea. 2008; 27: S1-S83Crossref PubMed Scopus (268) Google Scholar, 13Mashima Y. Imamura Y. Konishi M. Nagasawa A. Yamada M. Oguchi Y. Kudoh J. Shimizu N. Homogeneity of kerato-epithelin codon 124 mutations in Japanese patients with either of two types of corneal stromal dystrophy.Am. J. Hum. Genet. 1997; 61: 1448-1450Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Okada M. Yamamoto S. Watanabe H. Inoue Y. Tsujikawa M. Maeda N. Shimomura Y. Nishida K. Kinoshita S. Tano Y. Granular corneal dystrophy with homozygous mutations in the kerato-epithelin gene.Am. J. Ophthalmol. 1998; 126: 169-176Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). More than 30 different mutations in TGFBIp resulting in corneal dystrophies have been identified. Prominent among these are two mutational hotspots, namely Arg-124 and Arg-555 (7Kannabiran C. Klintworth G.K. TGFBI gene mutations in corneal dystrophies.Hum. Mutat. 2006; 27: 615-625Crossref PubMed Scopus (125) Google Scholar, 15Korvatska E. Munier F.L. Djemaï A. Wang M.X. Frueh B. Chiou A.G. Uffer S. Ballestrazzi E. Braunstein R.E. Forster R.K. Culbertson W.W. Boman H. Zografos L. Schorderet D.F. Mutation hot spots in 5q31-linked corneal dystrophies.Am. J. Hum. Genet. 1998; 62: 320-324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Mutations in these two locations comprise ∼50% of all TGFBIp-linked corneal dystrophies despite large geographical variations in their prevalence (16Munier F.L. Frueh B.E. Othenin-Girard P. Uffer S. Cousin P. Wang M.X. Héon E. Black G.C. Blasi M.A. Balestrazzi E. Lorenz B. Escoto R. Barraquer R. Hoeltzenbein M. Gloor B. Fossarello M. Singh A.D. Arsenijevic Y. Zografos L. Schorderet D.F. BIGH3 mutation spectrum in corneal dystrophies.Invest. Ophthalmol. Vis. Sci. 2002; 43: 949-954PubMed Google Scholar, 17Fujiki K. Nakayasu K. Kanai A. Corneal dystrophies in Japan.J. Hum. Genet. 2001; 46: 431-435Crossref PubMed Scopus (55) Google Scholar, 18Chakravarthi S.V. Kannabiran C. Sridhar M.S. Vemuganti G.K. TGFBI gene mutations causing lattice and granular corneal dystrophies in Indian patients.Invest Ophthalmol. Vis. Sci. 2005; 46: 121-125Crossref PubMed Scopus (58) Google Scholar). The remaining mutations causing TGFBIp-linked corneal dystrophy are all in the fourth FAS1 domain (FAS1–4) (7Kannabiran C. Klintworth G.K. TGFBI gene mutations in corneal dystrophies.Hum. Mutat. 2006; 27: 615-625Crossref PubMed Scopus (125) Google Scholar). The stability of full-length TGFBIp is mirrored by FAS1–4; destabilizing mutations in FAS1–4 destabilize the full-length protein to a comparable degree (19Runager K. Basaiawmoit R.V. Deva T. Andreasen M. Valnickova Z. Sørensen C.S. Karring H. Thøgersen I.B. Christiansen G. Underhaug J. Kristensen T. Nielsen N.C. Klintworth G.K. Otzen D.E. Enghild J.J. Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.J. Biol. Chem. 2011; 286: 4951-4958Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, FAS1–4 can be considered a good model system for full-length TGFBIp. TGFBIp is the major part of the protein inclusions found in the cornea, and abnormal protein turnover of the protein is associated with various mutations (20Streeten B.W. Qi Y. Klintworth G.K. Eagle Jr., R.C. Strauss J.A. Bennett K. Immunolocalization of β ig-h3 protein in 5q31-linked corneal dystrophies and normal corneas.Arch. Ophthalmol. 1999; 117: 67-75Crossref PubMed Scopus (101) Google Scholar, 21Korvatska E. Henry H. Mashima Y. Yamada M. Bachmann C. Munier F.L. Schorderet D.F. Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.J. Biol. Chem. 2000; 275: 11465-11469Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, no deposits of TGFBIp aggregates have been observed elsewhere in the human body. This has been suggested to be linked to the slow turnover of proteins in the cornea due to a lack of blood vessels (22Beebe D.C. Maintaining transparency. A review of the developmental physiology and pathophysiology of two avascular tissues.Semin. Cell Dev. Biol. 2008; 19: 125-133Crossref PubMed Scopus (76) Google Scholar). Furthermore, a C-terminal fragment of TGFBIp derived from FAS1–4 has been found to accumulate in amyloids of the mutant V624M, forming lattice-type deposits in the cornea (23Karring H. Runager K. Thøgersen I.B. Klintworth G.K. Højrup P. Enghild J.J. Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene.Exp. Eye Res. 2012; 96: 163-170Crossref PubMed Scopus (46) Google Scholar). The molecular events leading to protein aggregation and accumulation involved in corneal dystrophy are still unclear. The large variety of mutations in TGFBIp leading to different forms of corneal dystrophies indicates that TGFBIp may aggregate in a number of different ways. To probe this aggregate variability in more detail, we present a biophysical analysis of the aggregation of the FAS1–4 domain containing the naturally occurring disease-promoting amino acid substitution A546T. This mutation results in lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma and has an age of onset of 35–40 years (24Dighiero P. Drunat S. Ellies P. D'Hermies F. Savoldelli M. Legeais J.M. Renard G. Delpech M. Grateau G. Valleix S. A new mutation (A546T) of the β ig-h3 gene responsible for a French lattice corneal dystrophy type IIIA.Am. J. Ophthalmol. 2000; 129: 248-251Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We show that the A546T substitution, already known to destabilize FAS1–4 (19Runager K. Basaiawmoit R.V. Deva T. Andreasen M. Valnickova Z. Sørensen C.S. Karring H. Thøgersen I.B. Christiansen G. Underhaug J. Kristensen T. Nielsen N.C. Klintworth G.K. Otzen D.E. Enghild J.J. Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.J. Biol. Chem. 2011; 286: 4951-4958Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), leads to an increase in surface exposure of hydrophobic patches. The mutation also leads to the formation of two distinct fibril morphologies, namely long straight fibrils with pure β-sheet structure (formed at lower concentrations) and short and curly fibrils with a mixture of α-helical and β-sheet structures (formed at higher concentrations). Different oligomeric intermediates displaying different membrane permeabilization potential accumulate during the two distinct fibrillation pathways. We suggest the different oligomeric states found under the two different conditions give rise to the different fibril types. The complexity of the aggregation process induced by a single mutation provides a good indication that TGFBIp has access to a number of different aggregation pathways. All chemicals were purchased from Sigma. This work is part of our ongoing efforts to find optimal conditions for preparation of homogeneous fibrils for solid-state NMR elucidation of the molecular structure of these fibrils. Consequently, all fibrillation work in this study was carried out on 13C- and 15N-labeled FAS1–4 A546T. For chemical and thermal stability studies and the analysis of trypsin proteolysis, we used non-labeled FAS1–4 WT as a reference. Non-labeled FAS1–4 A546T was included as a control for the possible effect of using labeled protein instead of non-labeled protein. A546T FAS1–4 (residues 502–657) was cloned into a pET SUMO expression vector as described previously (19Runager K. Basaiawmoit R.V. Deva T. Andreasen M. Valnickova Z. Sørensen C.S. Karring H. Thøgersen I.B. Christiansen G. Underhaug J. Kristensen T. Nielsen N.C. Klintworth G.K. Otzen D.E. Enghild J.J. Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.J. Biol. Chem. 2011; 286: 4951-4958Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Escherichia coli BL21(DE3) cells were transformed with SUMO-FAS1–4 A546T plasmid (residue 502–657) plated on LB/kanamycin agar and left at 37 °C overnight. 2 ml of LB/kanamycin was inoculated with one colony and incubated overnight. The overnight culture was once again plated out and left at 37 °C overnight. One colony from this plate was used for protein expression in a fermenter after a pilot expression confirmed the expected size and sequence of the protein. 13C,15N-Labeled FAS1–4 A546T was expressed in a fermenter (type C 10–3, from B. Braun Biotech, Mesungen, Germany) in 8 liters of M9 medium using the same BL21(DE3) clone as described above. Protein expression was induced using 1 mm isopropyl 1-thio-β-d-galactopyranoside at which point M9 medium with d-[13C6]glucose and [15N]NH4Cl as carbon and nitrogen sources, respectively, was added. Protein expression was allowed to proceed for 10 h, after which cells were harvested by centrifugation. Frozen bacterial pellets from fermenter expression (batches of 10–20 g) were resuspended in 500 mm NaCl, 50 mm Tris-HCl, pH 7.6 (buffer A), sonicated for 6 × 30 s while on ice, and centrifuged for 10 min at 10,000 × g. The supernatant was then applied to a nickel column (5 ml HiTrap Chelating HP, GE Healthcare) pre-equilibrated with buffer A at 1–2 ml/min. The column was then washed with 3 column volumes of buffer A and attached to an Äkta Purifier (GE Healthcare), and bound protein was eluted with imidazole using a stepwise gradient of 500 mm NaCl, 200 mm imidazole, and 50 mm Tris-HCl, pH 7.6 (buffer B). Fractions containing SUMO-FAS1–4 fusion protein were pooled and dialyzed against 100 mm NaCl, 50 mm Tris-HCl, pH 7.6, before the fusion protein was cleaved by incubation for 24 h at 16 °C with 2 units/ml SUMO protease. After the cleaved protein was dialyzed against 2 × 5 liters of phosphate-buffered saline at 4 °C, the pure FAS1–4 A546T product was obtained as the flow-through from a nickel-nitrilotriacetic acid column. Finally, the purified protein was concentrated (YM CentriPrep, molecular weight cutoff 3 kDa) to a stock of 1.5 mg/ml. The protein was fibrillated in 20 mm phosphate buffer, pH 7.4, 20 mm NaCl at 0.4, 0.8, and 1.2 mg/ml protein. Additional NaCl or heparin was added to different concentrations. Thioflavin T (ThT) was added to the protein solution to a final concentration of 40 μm, and the protein solution was transferred to a 96-well black Costar polystyrene microtiter plate, sealed to prevent evaporation, and placed in an Infinite M200 plate reader (Tecan Nordic AB). When fibril seeds were present, these were added immediately before the plate was sealed. Seeds of preformed fibrils were produced by sonication for 10 s at 30% amplitude with a Bandelin Sonopuls HD 2070 sonication probe (Buch & Holm). The plate was incubated at 37 °C, and the ThT fluorescence (excitation 450 nm, emission 482 nm) was measured every 5 min with 3 min of shaking between each reading. The concentration of FAS1–4 A546T in the supernatant after fibrillation and centrifugation for 30 min at 13,000 rpm in a table top centrifuge was analyzed using a Bradford reagent kit (Sigma) with a standard curve of bovine serum albumin according to the manufacturer's recommendations. 0.4 mg/ml protein was incubated with 1, 0.1, 0.01, 0.001, and 0% trypsin (w/w %) for 30 min at 37 °C and then analyzed by reducing 15% SDS-PAGE. After Coomassie staining and destaining, the gel was scanned, and the intensities of the bands were determined using the software ImageJ. 0.17 mg/ml FAS1–4 was mixed with 10 μm ANS, and the fluorescence emission was measured from 400 to 600 nm with excitation at 365 nm, slit widths of 10 nm, and a scan speed of 200 nm/min on an LS55 luminescence spectrophotometer (PerkinElmer Life Sciences). Three spectra were accumulated and averaged for each sample. Fibril samples were sonicated for 2 s at 60% amplitude with a Bandelin probe before analysis to minimize light scattering (25Ghodke S. Nielsen S.B. Christiansen G. Hjuler H.A. Flink J. Otzen D. Mapping out the multistage fibrillation of glucagon.FEBS J. 2012; 279: 752-765Crossref PubMed Scopus (31) Google Scholar, 26Rakhit R. Cunningham P. Furtos-Matei A. Dahan S. Qi X.F. Crow J.P. Cashman N.R. Kondejewski L.H. Chakrabartty A. Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis.J. Biol. Chem. 2002; 277: 47551-47556Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). CD wavelength spectra from 250 to 200 nm with a step size of 0.2 nm, bandwidth of 2 nm, and scan speed of 50 nm/min were recorded at 25 °C with a J-810 CD-spectrometer (Jasco) using a 1-mm quartz cuvette (Hellma). Five spectra were averaged for each sample, and the buffer spectra were subtracted. CD thermal scans from 20 to 95 °C with a step size of 0.2 °C, bandwidth of 2 nm, and scan speed of 90 °C/h were recorded, and 3 scans were averaged for each sample. The obtained data points were fitted as described previously (27Mogensen J.E. Ipsen H. Holm J. Otzen D.E. Elimination of a misfolded folding intermediate by a single point mutation.Biochemistry. 2004; 43: 3357-3367Crossref PubMed Scopus (40) Google Scholar) using the software KaleidaGraph. FTIR spectroscopy was performed using a Tensor27 FTIR spectrometer (Bruker) equipped with attenuated total reflection accessory with a continuous flow of N2 gas. All samples were dried with N2 gas. 64 interferograms were accumulated at a spectral resolution of 2 cm−1. Peak positions were assigned where the second order derivative had local minima, and the intensity was modeled by Gaussian curve fitting using the OPUS 5.5 software. 0.17 mg/ml FAS1–4 A546T was mixed with various concentrations of urea and allowed to equilibrate for at least 2 h. A far-UV CD spectrum was acquired, and the data were fitted as described (27Mogensen J.E. Ipsen H. Holm J. Otzen D.E. Elimination of a misfolded folding intermediate by a single point mutation.Biochemistry. 2004; 43: 3357-3367Crossref PubMed Scopus (40) Google Scholar) using KaleidaGraph 4.0 (GeoMEM Consultants). Aliquots of 5 μl of fibril solution were mounted on 400-mesh carbon-coated, glow-discharged nickel grids for 30 s. The grids were washed with one drop of double distilled water and stained with three drops of 1% phosphotungstic acid, pH 7.2. Samples were inspected in a JEOL 1010 transmission electron microscope at 60 keV. Images were obtained using an electron sensitive Olympus KeenView CCD camera. The development of individual aggregation species during the course of FAS1–4 A546T fibrillations at 0.4 and 1.2 mg/ml was monitored using a Postnova AF2000 asymmetrical AF4 system (Postnova Analytics GmbH) consisting of two HPLC pumps, a AF2000 focus and separation unit equipped with a 10-kDa molecular weight cutoff ultrafiltration membrane, and a 350-μm spacer defining the trapezoidal shape of the separation channel, a S3240 UV-visible detector set at a wave length of 205 nm, and a PN3140 refractive index detector. Samples were centrifuged at 13,000 × g for 5 min before analysis to remove insoluble material. 60 μg of FAS1–4 (50 μl of 1.2 mg/ml or 150 μl of 0.4 mg/ml) was injected using a glass syringe into an appropriate sample loop mounted in a Rheodyne injection port. The sample was injected and separated in 20 mm phosphate buffer, pH 7.4, 20 mm NaCl using the following flow program modified from Lorenzen et al. (28Lorenzen N. Cohen S.I. Nielsen S.B. Herling T.W. Christiansen G. Dobson C.M. Knowles T.P. Otzen D. Role of elongation and secondary pathways in s6 amyloid fibril growth.Biophys. J. 2012; 102: 2167-2175Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A constant detector/outlet flow of 0.5 ml/min was maintained throughout the separation: 1) sample loading using an injection/tip flow of 0.2 ml/min, cross-flow of 2.5 ml/min, and focus flow of 2.8 ml/min; 2) a 1-min linear gradient to 3 ml/min tip-flow and 2.5 ml/min cross-flow (initiation of elution); 3) isocratic elution of low Mr species (e.g. FAS1–4 monomer, dimer, and trimer) at 3 ml/min tip-flow and 2.5 ml/min cross-flow; 4) a 25-min linear cross-flow gradient to 0.25 ml/min; 5) a 15-min linear cross-flow gradient to 0 ml/min (field release; no separation); 6) a 10-min washing/rinsing step at 0 ml/min cross-flow. The concentration of eluting species was monitored by absorbance at 205 nm because of the low content of aromatic residues in FAS1–4 (precluding the use of absorption at 280 nm) and artifacts from the refractive index signals generated when applying the cross-flow gradients. The correlation between the retention time and the apparent molecular weight was approximated based on a calibration curve made with ribonuclease A (13.7 kDa), carbonic anhydrase (29/58 kDa), aldolase (158 kDa), and thyroglobulin (669/1338 kDa) (all from GE Healthcare gel filtration low and high molecular weight calibration kits) by plotting the retention time against the molecular weight. Calcein vesicles were prepared by dissolving 5 mg/ml lipids of either 100% 1,2-dioleoyl-sn-[phosphor-rac-(1-glycerol)] (DOPG), 100% 1,2-dioleoyl-sn-glycero-3-phospho-choline (DOPC), or 50% DOPG:50% DOPC in chloroform. Chloroform was evaporated, and the lipids were dissolved in 70 μm calcein, 20 mm NaPi, pH 7.4, and 20 mm NaCl. The lipids were flash-frozen in liquid nitrogen and thawed to produce unilamellar vesicles. The lipid vesicles were extruded through a 100-μm filter (Avanti Polar Lipids) to ensure a uniform size of the vesicles. 600 μl of calcein vesicles were eluted with 1.5 ml 20 mm NaPi, pH 7.4, and 20 mm NaCl from a PD10 desalting column equilibrated in 20 mm NaPi, pH 7.4, 20 mm NaCl. Samples removed from fibrillating FAS1–4 A546T at 1.2 and 0.4 mg/ml 0.001% heparin were centrifuged at 13,000 × g in a tabletop centrifuge. The supernatant was removed, and the sample was diluted to 0.02 mg/ml protein by the addition of 20 mm NaPi, pH 7.4, 20 mm NaCl. Nine additional steps of 2-fold dilutions were prepared. 150 μl of each dilution was transferred to a 96-well black Costar polystyrene microtiter plate. The plate was placed in an Infinite M200 plate reader at 37 °C. The fluorescence with excitation at 485 nm and emission at 520 nm was measured with 5 s of shaking between each read. After 10 min the plate was removed, and 2 μl of 5 mg/ml lipid calcein vesicles were added. The plate was placed in the Infinite M200 plate reader at 37 °C. Fluorescence emission intensity (excitation at 485 nm and emission at 520 nm) was recorded with 5 s of shaking between each read. After ∼1 h, the plate was removed, and 2 μl of 2% Triton X-100 was added to each well to allow complete lysis of vesicles followed by emission recording as above. We initially compared the stability of the non-fibrillated FAS1–4 A546T relative to FAS1–4 WT by monitoring chemical and thermal unfolding by CD spectroscopy. This was done by following the changes in the intensity at 222 nm as a function of either chemical denaturant concentration or temperature. FAS1–4 A546T was less stable than FAS1–4 WT both with regard to chemical and thermal denaturation (Fig. 1, A and B). Melting temperatures (Tm) were 52.3 °C for FAS1–4 A546T versus 66.8 °C for WT, whereas the midpoints of denaturation ([urea]50%) were 1.78 and 3.07 m urea for A546T and WT, respectively (Table 1). The free energy of unfolding (ΔGD-N) of A546T are less than half that of WT, consistent with results previously obtained for unlabeled FAS1–4 mutants (19Runager K. Basaiawmoit R.V. Deva T. Andreasen M. Valnickova Z. Sørensen C.S. Karring H. Thøgersen I.B. Christiansen G. Underhaug J. Kristensen T. Nielsen N.C. Klintworth G.K. Otzen D.E. Enghild J.J. Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.J. Biol. Chem. 2011; 286: 4951-4958Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, as expected, isotopic labeling of FAS1–4 does not change protein stability. The α-helical and β-sheet contributions to the secondary structure of FAS1–4 WT and A546T were estimated by spectral deconvolution of the far-UV CD spectra (Fig. 1A, inset) using the K2d algorithm (29Andrade M.A. Chacón P. Merelo J.J. Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network.Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (949) Google Scholar). The β-sheet content of both FAS1–4 WT and A546T was estimated to be 16%, whereas the α-helical contribution was estimated to be 28% for WT and 27% for A546T. This distribution correlates with what has previously been reported for Fas1–4 (10Clout N.J. Tisi D. Hohenester E. Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I.Structure. 2003; 11: 197-203Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). We conclude that the labeling and the amino acid substitution do not change the structure of the monomeric protein (19Runager K. Basaiawmoit R.V. Deva T. Andreasen M. Valnickova Z. Sørensen C.S. Karring H. Thøgersen I.B. Christiansen G. Underhaug J. Kristensen T. Nielsen N.C. Klintworth G.K. Otzen D.E. Enghild J.J. Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.J. Biol. Chem. 20" @default.
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• W2068918178 title "Polymorphic Fibrillation of the Destabilized Fourth Fasciclin-1 Domain Mutant A546T of the Transforming Growth Factor-β-induced Protein (TGFBIp) Occurs through Multiple Pathways with Different Oligomeric Intermediates" @default.
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