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• W2025972448 abstract "Mg2+-induced polymerization of type III intermediate filament proteins vimentin and glial fibrillary acidic protein was studied by transient electric birefringence. In the absence of MgCl2 we found a net permanent dipole moment, ∼45-nm-long dimers for vimentin, ∼65-nm-long tetramers, hexamers, and possibly octamers for both proteins, and 100-nm aggregates for glial fibrillary acidic protein. Controlled oligomerization occurred after the addition of MgCl2. Although the solutions contained (small) aggregates of different sizes, more or less discrete steps in polymer formation were observed, and it was possible to discriminate between an increase in width and length. At the first stage of polymerization (in 0.3 mm MgCl2 for vimentin and 0.2 mm MgCl2 for glial fibrillary acidic protein), the permanent dipole moment disappeared without a change in length of the particles. At higher MgCl2concentrations, structures of approximately 100 nm were formed, which strongly tended to laterally assemble into full-width intermediate filament structures consisting of about 32 monomers. This contrasts with previous models where first full-width (∼10-nm) aggregates are formed, which then increase in length. Subsequently, two discrete elongation steps of 35 nm are observed that increase the length to 135 and 170 nm, respectively. Possible structural models are suggested for the polymerization. Mg2+-induced polymerization of type III intermediate filament proteins vimentin and glial fibrillary acidic protein was studied by transient electric birefringence. In the absence of MgCl2 we found a net permanent dipole moment, ∼45-nm-long dimers for vimentin, ∼65-nm-long tetramers, hexamers, and possibly octamers for both proteins, and 100-nm aggregates for glial fibrillary acidic protein. Controlled oligomerization occurred after the addition of MgCl2. Although the solutions contained (small) aggregates of different sizes, more or less discrete steps in polymer formation were observed, and it was possible to discriminate between an increase in width and length. At the first stage of polymerization (in 0.3 mm MgCl2 for vimentin and 0.2 mm MgCl2 for glial fibrillary acidic protein), the permanent dipole moment disappeared without a change in length of the particles. At higher MgCl2concentrations, structures of approximately 100 nm were formed, which strongly tended to laterally assemble into full-width intermediate filament structures consisting of about 32 monomers. This contrasts with previous models where first full-width (∼10-nm) aggregates are formed, which then increase in length. Subsequently, two discrete elongation steps of 35 nm are observed that increase the length to 135 and 170 nm, respectively. Possible structural models are suggested for the polymerization. Many eukaryotic cells contain cytoplasmic intermediate filaments (IFs), 1The abbreviations used are: IF, intermediate filaments; TEB, transient electric birefringence; GFAP, glial fibrillary acidic protein; Ω, ohm; SI, sample interval. which are composed of proteins with a molecular mass of 40–210 kDa (1Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1284) Google Scholar, 2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar). All IF proteins contain a central α-helix-rich region, consisting of approximately 310 amino acids, flanked by nonhelical amino- and carboxy-end domains, which are extremely diverse in size, composition, and charge characteristics. The α-helical regions of two IF protein chains assemble parallel and exactly in register to form a 45-nm coiled-coil rod with end domains of uncertain length (2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar, 3Quinlan R.A. Franke W.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 97: 3452-3456Crossref Scopus (112) Google Scholar, 4Kaufmann E. Weber K. Geisler N. J. Mol. Biol. 1985; 185: 733-742Crossref PubMed Scopus (125) Google Scholar). In a recent publication on vimentin IF structures, we have proven the occurrence of such dimers in low ionic strength solution (5Quinlan R.A. Hatzfeld M. Franke W.W. Lustig A. Schulthess T. Engel J. J. Mol. Biol. 1986; 192: 337-349Crossref PubMed Scopus (86) Google Scholar). We have also shown that these rods are bent and/or flexible structures (7Kooijman M. van Amerongen H. Traub P. van Grondelle R. Bloemendal M. FEBS Lett. 1995; 358: 185-188Crossref PubMed Scopus (12) Google Scholar). In the late eighties and early nineties, much work was done to elucidate the next level of IF organization (5Quinlan R.A. Hatzfeld M. Franke W.W. Lustig A. Schulthess T. Engel J. J. Mol. Biol. 1986; 192: 337-349Crossref PubMed Scopus (86) Google Scholar, 8Ip W. Hartzer M.K. Susana Pang Y.-Y Robson R.M. J. Mol. Biol. 1985; 183: 365-375Crossref PubMed Scopus (111) Google Scholar, 9Potschka M. Nave R. Weber K. Geisler N. Eur. J. Biochem. 1990; 190: 503-508Crossref PubMed Scopus (25) Google Scholar, 10Steinert P.M. J. Struct. Biol. 1991; 107: 157-174Crossref PubMed Scopus (53) Google Scholar, 11Steinert P.M. J. Struct. Biol. 1991; 107: 175-188Crossref PubMed Scopus (63) Google Scholar, 12Geisler N. Schünemann J. Weber K. Eur. J. Biochem. 1992; 206: 841-852Crossref PubMed Scopus (87) Google Scholar, 13Aebi U. Fowler W.E. Rew P. Sun T.-T. J. Cell. Biol. 1983; 97: 1131-1143Crossref PubMed Scopus (155) Google Scholar, 14Aebi U. Häner M. Troncoso J. Eichner R. Engel A. Protoplasma. 1988; 145: 73-81Crossref Scopus (105) Google Scholar, 15Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 16Eichner R. Rew P. Engel A. Aebi U. Annu. N. Y. Acad. Sci. 1985; 455: 381-402Crossref PubMed Scopus (36) Google Scholar, 17Geisler N. FEBS Lett. 1993; 323: 63-67Crossref PubMed Scopus (12) Google Scholar, 18Steinert P.M. Parry D.A.D. J. Biol. Chem. 1993; 268: 2878-2887Abstract Full Text PDF PubMed Google Scholar, 19Steinert P.M. Marekow L.N. Fraser R.D.B. Parry D.A.D. J. Mol. Biol. 1993; 230: 436-452Crossref PubMed Scopus (239) Google Scholar, 20Steinert P.M. Marekow L.N. Parry D.A.D. Biochemistry. 1993; 32: 10046-10056Crossref PubMed Scopus (58) Google Scholar, 21Steinert P.M. Marekow L.N. Parry D.A.D. J. Biol. Chem. 1993; 268: 24916-24925Abstract Full Text PDF PubMed Google Scholar, 22Heins S. Wong P.C. Müller S.A. Goldie K. Cleveland D.W. Aebi U. J. Cell. Biol. 1993; 123: 1517-1533Crossref PubMed Scopus (142) Google Scholar, 23Parry D.A.D. Proteins Struct. Funct. Genet. 1995; 22: 267-272Crossref PubMed Scopus (25) Google Scholar). Most of these studies have been reviewed by Heins and Aebi (2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar). At the level of tetramers, essentially four alignments of the constituting dimers have been suggested: antiparallel in register forming a rod approximately 45 nm long, head-to-tail with slight overlap and antiparallel staggered in two different ways, yielding a rod length of approximately 65 nm. Together with other data (9Potschka M. Nave R. Weber K. Geisler N. Eur. J. Biochem. 1990; 190: 503-508Crossref PubMed Scopus (25) Google Scholar, 10Steinert P.M. J. Struct. Biol. 1991; 107: 157-174Crossref PubMed Scopus (53) Google Scholar, 11Steinert P.M. J. Struct. Biol. 1991; 107: 175-188Crossref PubMed Scopus (63) Google Scholar), our transient electric birefringence (TEB) data on vimentin missing the first 70 amino acids (T-vimentin) (24Kooijman M. Bloemendal M. Traub P. van Grondelle R. van Amerongen H. J. Biol. Chem. 1995; 270: 2931-2937Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), intact vimentin (6Kooijman M. Bloemendal M. van Amerongen H. Traub P. van Grondelle R. J. Mol. Biol. 1994; 236: 1241-1249Crossref PubMed Scopus (13) Google Scholar), desmin, and glial fibrillary acidic protein (GFAP) (7Kooijman M. van Amerongen H. Traub P. van Grondelle R. Bloemendal M. FEBS Lett. 1995; 358: 185-188Crossref PubMed Scopus (12) Google Scholar) provided evidence for the predominant occurrence of the latter. Moreover, we detected particles with a length very similar to that of staggered tetramers but with a much larger permanent dipole moment (6Kooijman M. Bloemendal M. van Amerongen H. Traub P. van Grondelle R. J. Mol. Biol. 1994; 236: 1241-1249Crossref PubMed Scopus (13) Google Scholar, 7Kooijman M. van Amerongen H. Traub P. van Grondelle R. Bloemendal M. FEBS Lett. 1995; 358: 185-188Crossref PubMed Scopus (12) Google Scholar). Most likely, these represent hexamers as suggested by Steinert (10Steinert P.M. J. Struct. Biol. 1991; 107: 157-174Crossref PubMed Scopus (53) Google Scholar, 11Steinert P.M. J. Struct. Biol. 1991; 107: 175-188Crossref PubMed Scopus (63) Google Scholar) and Herrmann et al. (15Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar). Conflicting models for the molecular architecture of higher oligomers have been suggested (2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar, 12Geisler N. Schünemann J. Weber K. Eur. J. Biochem. 1992; 206: 841-852Crossref PubMed Scopus (87) Google Scholar, 19Steinert P.M. Marekow L.N. Fraser R.D.B. Parry D.A.D. J. Mol. Biol. 1993; 230: 436-452Crossref PubMed Scopus (239) Google Scholar, 21Steinert P.M. Marekow L.N. Parry D.A.D. J. Biol. Chem. 1993; 268: 24916-24925Abstract Full Text PDF PubMed Google Scholar), and intermediate steps in filament formation are not well understood (17Geisler N. FEBS Lett. 1993; 323: 63-67Crossref PubMed Scopus (12) Google Scholar, 21Steinert P.M. Marekow L.N. Parry D.A.D. J. Biol. Chem. 1993; 268: 24916-24925Abstract Full Text PDF PubMed Google Scholar). As mentioned by Heins and Aebi (2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar) “the hierarchy of the three types of lateral dimer-dimer interaction is ambiguous in these models.” Furthermore, it is not clear what the exact number of IF monomers in filament cross-section is, this may be varying in different filaments and even within the same filament (2Heins S. Aebi U. Curr. Opin. Cell. Biol. 1994; 6: 25-33Crossref PubMed Scopus (105) Google Scholar, 22Heins S. Wong P.C. Müller S.A. Goldie K. Cleveland D.W. Aebi U. J. Cell. Biol. 1993; 123: 1517-1533Crossref PubMed Scopus (142) Google Scholar, 25Herrmann H. Häner M. Brettel M. Müller S.A. Goldie K.N. Fedtke B. Lustig A. Franke W.W. Aebi U. J. Mol. Biol. 1996; 264: 933-953Crossref PubMed Scopus (271) Google Scholar). Most evidence for current ideas on IF assembly is based on EM results and cross-linking studies. The latter give very detailed information on the connections between amino acids of different protein subunits, but the hierarchy of dimer-dimer interactions in IFs cannot be deduced from them, the size distribution of the oligomers is unknown, and direct evidence for higher structures is not obtained. To verify postulated models in solution and to obtain more information on three-dimensional growth and organization of IFs, we have performed a TEB study on MgCl2-induced filament formation of the type III IF proteins vimentin and GFAP. To our knowledge, this is the first detailed study on the architecture of complete glial filaments, the paper by Stewart et al. (26Stewart M. Quinlan R.A. Moyer R.D. J. Cell Biol. 1989; 109: 225-234Crossref PubMed Scopus (55) Google Scholar) on the rod portion of GFAP being the only other detailed structural study on this protein. Vimentin from Ehrlich ascites tumor cells and GFAP from bovine brain white matter, purified as described (27Vorgias C.E. Traub P. Biochem. Biophys. Res. Commun. 1983; 115: 68-75Crossref PubMed Scopus (19) Google Scholar), lyophilized, and stored at −20 °C, was dissolved in 2 mm phosphate, pH 7.5, 6 mm 2-mercaptoethanol, 6 m urea. From this buffer, the proteins were dialyzed at 4 °C for at least 16 h against 0.7 mm sodium phosphate, pH 7.5, 6 mm2-mercaptoethanol (vimentin), and 2 mm BisTris·HCl, pH 6.8, 6 mm 2-mercaptoethanol (GFAP). The vimentin and GFAP concentrations in the absence of MgCl2 were determined from the absorption using a molar extinction coefficient (ε) (per polypeptide chain) at 280 nm of 24,240 m−1cm−1 for vimentin and 20,100 m−1cm−1 for GFAP, calculated from the extinction coefficients of the individual tryptophan (ε = 6300 m−1cm−1) and tyrosine (ε = 1380m−1 cm−1) residues (28). Protein concentrations were adjusted to 0.08–0.14 mg/ml. When dissolved optically anisotropic molecules are oriented by a square electric-field pulse, the solution becomes birefringent. When the field is switched off, the order and hence the birefringence decays by rotational diffusion with a rate that depends on the size and the shape of the molecules. From the accurately measurable time-resolved birefringence, the following parameters can be determined. 1) The ratio of the areas enclosed by the rise and decay curve of the birefringence, S 1/S 2 (see Fig. 1), which is related to the relative magnitudes of the permanent (P) and induced (Q) dipole moments of the particles according to Ref. 29Fredericq E. Houssier C. Electric Dichroism and Electric Birefringence.in: Harrington W. Peacocke A.R. Clarendon Press, Oxford1973Google Scholar.S1S2=4PQ+1/PQ+1Equation 1 2) The Kerr constant (K), which is defined as the proportionality constant between the magnitude of the steady-state birefringence, Δn 0, and the square of the electric field E (K = Δn 0/E 2) at low fields;K is proportional to the product of P +Q and the optical anisotropy factor of the molecule. 3) The field-free decay time τ of the birefringence signal, yielding information on size and flexibility of the molecules (29Fredericq E. Houssier C. Electric Dichroism and Electric Birefringence.in: Harrington W. Peacocke A.R. Clarendon Press, Oxford1973Google Scholar). In a multispecies system, the average decay time τav is given by the equation,τav=∑iciKiτi∑iciKiEquation 2 where c i and K i are the concentration and Kerr constant of species i with decay time τ i. Here we treat the filament aggregates as rigid cylindrical rods. For such particles, τ equals 1/(6D r), whereD r is the end-over-end rotational diffusion coefficient (s−1). Several expressions forD r have been developed (30Broersma S. J. Chem. Phys. 1960; 32: 1626-1631Crossref Scopus (570) Google Scholar, 31Broersma S. J. Chem. Phys. 1981; 74: 6989-6990Crossref Scopus (173) Google Scholar, 32Mandelkern M. Elias J.G. Eden D. Crothers D.M. J. Mol. Biol. 1981; 152: 153-161Crossref PubMed Scopus (171) Google Scholar).D r is strongly dependent on the length and rather insensitive to changes in the diameter. The electric birefringence setup has been described in detail (33Van Haeringen B. Jiskoot W. van Grondelle R. Bloemendal M. J. Biomol. Struct. & Dyn. 1992; 9: 991-1011Crossref PubMed Scopus (9) Google Scholar). The field strength was varied between 0.3 and 4.0 kV/cm, the pulse width between 70 and 500 μs, and the sample interval between 0.1 and 1.0 μs. At least one early experiment was repeated at the end of a series to ensure that the samples remained intact. For each birefringence curve, at least 100 transients were averaged. Field strengths were determined by averaging five electric field pulses. Measurements were performed at 20 °C. To study the assembly of GFAP and vimentin IF under the influence of MgCl2 (or NaCl), small aliquots of a 20 mmstock solution were added to the protein solution (volume 0.8 ml) and mixed using a Pasteur pipette. Transients were measured after 10 min and repeated until the TEB signal was constant. Conductivity of the samples was determined by measuring the impedance of the solution in the Kerr cell at 1 kHz (Fluke Impedance Bridge model 710B). The resistance of the solution ranged from 400 ± 100 Ω in the absence of MgCl2 to approximately 80 Ω at 2.5 mm MgCl2. The Kerr constants were determined in solutions with a resistance of 200 Ω by dialysis of the proteins from the aqueous low salt buffers (0.7 mm sodium phosphate, pH 7.5, and 2 mmBisTris·HCl, pH 6.8) to buffers with different concentrations of MgCl2 (see Table I). The resistance was then adjusted to 200 Ω with NaCl, which did not change the birefringence decay signals. For vimentin at 1 mm MgCl2, where the resistance of the solution was 140 Ω only, a correction of the Kerr constant to 200-Ω solutions was made. Since the Kerr constants of vimentin at pH 8.5 and T-vimentin at pH 7.5 (in the presence and absence of MgCl2) were approximately 30% smaller at 140 than at 200 Ω, the corrected K value for vimentin at 1 mm MgCl2 as presented in Table I is 50% larger than the measured one.Table IKerr constants for 1 m protein chain and S1/S2 values of vimentin and GFAP at different MgCl2 concentrationprotein[MgCl2]Pulse lengthS 1/S 2Kmmμs× 10 −13 m 2 V −2Vimentin0702.1 ± 0.11.2 ± 0.2Vimentin0.3701.1 ± 0.10.9 ± 0.2Vimentin0.52001.1 ± 0.14.0 ± 1.0Vimentin1.05001.1 ± 0.113.2 ± 1.31-aCorrected value (see “Experimental Procedures”).GFAP01401.5 ± 0.21.4 ± 0.3GFAP0.21401.2 ± 0.11.4 ± 0.3GFAP0.35001.1 ± 0.112.4 ± 0.8GFAP0.55001.1 ± 0.117.0 ± 1.1The resistance of the solution was adjusted to 200 Ω (see “Experimental Procedures”).1-a Corrected value (see “Experimental Procedures”). Open table in a new tab The resistance of the solution was adjusted to 200 Ω (see “Experimental Procedures”). Steady-state birefringence was determined by averaging the plateau of the rise curve. The field-free decay of the birefringence was analyzed on a SUN4 workstation using the computer programs DISCRETE (34Provencher S.W. Biophys. J. 1976; 16: 27-41Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 35Provencher S.W. J. Chem. Phys. 1976; 64: 2772-2777Crossref Scopus (621) Google Scholar) and CONTIN (36Provencher S.W. Comp. Phys. Commun. 1982; 27: 213-227Crossref Scopus (2623) Google Scholar, 37Provencher S.W. Comp. Phys. Commun. 1982; 27: 229-242Crossref Scopus (2549) Google Scholar). The former fits data with discrete exponential decays, while the latter uses a continuous distribution of exponential decay times. In this paper we predominantly present results obtained from the analysis with DISCRETE, but in all cases the data were also analyzed with CONTIN to ensure that consistent results were obtained. Analyses were started 0.20 μs (when the sample interval (SI) = 0.1 or 0.2 μs), 0.50 μs (SI = 0.5 μs), or 1.0 μs (SI = 1.0 μs) after the orienting field was turned off. The TEB signal of vimentin at pH 7.5 in the absence of MgCl2 is shown in Fig. 1. TheS 1/S 2 value (2.1 ± 0.1) is somewhat lower than that reported previously for vimentin at pH 6.8 and 8.5 (6Kooijman M. Bloemendal M. van Amerongen H. Traub P. van Grondelle R. J. Mol. Biol. 1994; 236: 1241-1249Crossref PubMed Scopus (13) Google Scholar), presumably due to the use of a slightly different buffer. When 0.3 mm MgCl2 is added,S 1/S 2 decreases from 2.1 ± 0.1 to 1.1 ± 0.1 (TableI), showing that the permanent dipole moment has almost disappeared. Such a decrease is not observed when MgCl2 is replaced by NaCl under conditions of equal sample resistance, reflecting a specific role of Mg2+. The Kerr constant of vimentin in the absence of MgCl2 is 1.2 ± 0.2 × 10−13 m2V−2 for 1m protein chain, a value comparable with that at pH 6.8 and 8.5 (6Kooijman M. Bloemendal M. van Amerongen H. Traub P. van Grondelle R. J. Mol. Biol. 1994; 236: 1241-1249Crossref PubMed Scopus (13) Google Scholar). The addition of 0.3 mm MgCl2 does not change the Kerr constant to a large extent (Table I). For GFAP at pH 6.8, the effect of small amounts of MgCl2 (0.2 mm) on S 1/S 2is comparable with that for vimentin (a decrease from 1.5 ± 0.2 to 1.2 ± 0.1; see Table I), while NaCl did not result in such a decrease. The Kerr constant of GFAP is not affected between 0 and 0.2 mm MgCl2 (Table I). Raising the MgCl2 concentration from 0.3 to 1.0 mm for vimentin and from 0.2 to 0.5 mm for GFAP leads to an increase of K by a factor of 15 and 12, respectively (Table I). On the other hand, theS 1/S 2 values of both proteins are not affected. At higher MgCl2 concentrations, no steady-state birefringence could be reached; thus, no Kerr constants and S 1/S 2 values could be estimated. CONTIN and DISCRETE analyses of the birefringence decay signals of vimentin (after 70-μs pulses) and GFAP (after 140-μs pulses) are presented in Tables II andIII. For clarity, the decay processes in the tables have been denoted τ1 … τ6(Table II) or τ1 … τ7 (Table III) with corresponding relative contributionsA 1 … A 6 orA 1 … A 7. This classification is just for convenience and does not automatically mean that, for instance, all τ3 values in the table are identical (compare τ3 of GFAP at 0.2 mm and 2.5 mm MgCl2). At MgCl2concentrations higher than 2.5 mm, CONTIN (showing a broad distribution of decay times) and DISCRETE are not able to resolve the decay processes adequately (data not shown). However, up to 2.5 mm MgCl2 well defined and mutually consistent decay times are produced by CONTIN and DISCRETE, CONTIN giving fully resolved distributions around the average decay times of DISCRETE. This is illustrated in Fig. 3 for GFAP at 2.5 MgCl2, where a good description requires decay times of approximately 40, 100, and 430 μs. Apparently, up to 2.5 mm MgCl2, a limited number of species are present in the solution.Table IITEB decay analysis after electric field pulses of vimentin at various concentrations of MgCl2 by CONTIN and DISCRETE[MgCl2]A 1τ1A 2τ2A 3τ3A 4τ4A 5τ5A 6τ6mm%μs%μs%μs%μs%μs%μs02-aSample interval was 0.1 μs.48 ± 51.2 ± 0.848 ± 55.3 ± 1.3<424 ± 80.32-aSample interval was 0.1 μs.52 ± 61.2 ± 0.844 ± 65.5 ± 1.5<630 ± 100.52-bSample interval was 0.5 μs.13 ± 41.4 ± 0.560 ± 85.5 ± 1.527 ± 1226 ± 51.02-bSample interval was 0.5 μs.<104.0 ± 2.075 ± 829 ± 825 ± 670 ± 171.42-bSample interval was 0.5 μs.35 ± 733 ± 755 ± 560 ± 10<10170 ± 501.82-bSample interval was 0.5 μs.60 ± 102-cDecay times not assigned to distinct oligomeric species but to a bent/flexible structure (see “Results”).44 ± 52-cDecay times not assigned to distinct oligomeric species but to a bent/flexible structure (see “Results”).35 ± 7120 ± 15<5250 ± 502.52-bSample interval was 0.5 μs.45 ± 82-cDecay times not assigned to distinct oligomeric species but to a bent/flexible structure (see “Results”).42 ± 62-cDecay times not assigned to distinct oligomeric species but to a bent/flexible structure (see “Results”).50 ± 8110 ± 15<5250 ± 50Decay times (τ) and their relative contributions (A) assigned to distinct oligomeric species with a relative contribution >10% are denoted in boldface type; decay times with a relative contribution <10% are denoted in italics. Pulse length was 70 μs.2-a Sample interval was 0.1 μs.2-b Sample interval was 0.5 μs.2-c Decay times not assigned to distinct oligomeric species but to a bent/flexible structure (see “Results”). Open table in a new tab Table IIITEB decay analysis of GFAP at various concentrations of MgCl2by CONTIN and DISCRETE[MgCl2]A 1τ1A 2τ2A 3τ3A 4τ4A 5τ5A 6τ6A 7τ7mm%μs%μs%μs%μs%μs%μs%μs03-aSample interval was 0.2 μs.35 ± 53-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).1.0 ± 0.43-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).45 ± 56.0 ± 1.020 ± 1017 ± 40.23-aSample interval was 0.2 μs.30 ± 73-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).1.0 ± 0.53-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).40 ± 55.5 ± 0.820 ± 817 ± 4<555 ± 150.33-aSample interval was 0.2 μs.70 ± 630 ± 330 ± 666 ± 5<2170 ± 500.53-cSample interval was 0.5 μs.52 ± 529 ± 545 ± 575 ± 8<3240 ± 501.03-cSample interval was 0.5 μs.67 ± 103-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).44 ± 53-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).30 ± 7120 ± 15<3400 ± 502.03-dSample interval was 1.0 μs.40 ± 53-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).40 ± 63-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).43 ± 4104 ± 1517 ± 9440 ± 302.53-dSample interval was 1.0 μs.25 ± 43-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).37 ± 33-bDecay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).61 ± 496 ± 814 ± 8430 ± 20Decay times (τ) and their relative contributions (A) assigned to distinct oligomeric species with a relative contribution >10% are denoted in boldface type; decay times with a relative contribution <10% are denoted in italics. Pulse length was 140 μs.3-a Sample interval was 0.2 μs.3-b Decay times not assigned to distinct oligomeric species but to a bend/flexible structure (see “Results”).3-c Sample interval was 0.5 μs.3-d Sample interval was 1.0 μs. Open table in a new tab Decay times (τ) and their relative contributions (A) assigned to distinct oligomeric species with a relative contribution >10% are denoted in boldface type; decay times with a relative contribution <10% are denoted in italics. Pulse length was 70 μs. Decay times (τ) and their relative contributions (A) assigned to distinct oligomeric species with a relative contribution >10% are denoted in boldface type; decay times with a relative contribution <10% are denoted in italics. Pulse length was 140 μs. For vimentin at 0 mm MgCl2, two major decay processes (1.2 ± 0.8 μs (τ1) and 5.3 ± 1.3 μs (τ2)), and a small contribution (<4%) of a longer component (24 ± 8 μs) (Table II) are found. In our previous studies on vimentin at pH 6.8 and 8.5 in the absence of MgCl2 (6Kooijman M. Bloemendal M. van Amerongen H. Traub P. van Grondelle R. J. Mol. Biol. 1994; 236: 1241-1249Crossref PubMed Scopus (13) Google Scholar, 24Kooijman M. Bloemendal M. Traub P. van Grondelle R. van Amerongen H. J. Biol. Chem. 1995; 270: 2931-2937Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), we found that decay times of 0.3–2.0 μs can be assigned to dimers of approximately 45 nm in length with a flexible and/or bent structure and that decay times of 4.0–7.0 μs can be assigned to staggered particles (probably tetramers and hexamers, possibly also octamers) with a length of 60–70 nm. Therefore, we conclude that, as at pH 6.8, vimentin at pH 7.5 without MgCl2 is mainly present as single dimers and staggered particles. At 0.3 mm MgCl2, the decay times are almost identical to those in the absence of MgCl2, suggesting that the average length of the particles does not change. Also for GFAP at 0 or 0.2 mm MgCl2, a decay time of 5.5 ± 1.5 μs is found (Table III). As for vimentin, we assign this to staggered particles (probably tetramers and hexamers, possibly also octamers) with a length of 60–70 nm. However, additionally, a small component with a decay time of 17 ± 4 μs is detected (Table III), showing that larger aggregates are already formed in the absence of MgCl2. The shortest decay time of GFAP (τ1 in Table III) has been described and analyzed before (7Kooijman M. van Amerongen H. Traub P. van Grondelle R. Bloemendal M. FEBS Lett. 1995; 358: 185-188Crossref PubMed Scopus (12) Google Scholar) and is probably not due to dimers but arises from a bent and/or flexible higher oligomer. This can explain the lower value forS 1/S 2 in comparison with vimentin, since fewer dimers means a lower permanent dipole moment contribution to the orientation mechanism of the particles. At higher MgCl2 concentrations, the average TEB decay time (τav) becomes much larger (see Fig.2), whereas addition of NaCl (not shown) had no effect on τav. For vimentin, formation of larger aggregates starts at 0.5 mmMgCl2. A “new” component with a decay time of 26 ± 5 μs (τ3) and a relative contribution of approximately 27% (see Table II) appears, but a significant fraction of dimers and staggered structures (τ1 and τ2) is still present. τ1 and τ2 disappear at higher concentrations of MgCl2, and a gradual shift to longer decay times due to larger particles (τ3, τ4, and τ5) is observed. At 1.8 and 2.5 mm MgCl2, two major decay times, around 40 and 110 μs, are found. The 40-μs component is intermediate between τ3 and τ4 as obtained at lower MgCl2 concentrations and might be an unresolved average of the two. Alternatively, it is due to a flexibility mode of higher oligomers. For GFAP, a similar pattern with almost identical decay times as for vimentin is observed (Table III, Fig. 2), but assembly occurs at lower MgCl2 concentrations. Larger structures are formed at 0.3 mm MgCl2, where τ1 and τ2 are no longer observed. Above 1.0 mmMgCl2, the shortest decay time (τ3) reflects either the average of τ3 and τ4 measured at lower MgCl2 concentrations or flexibility of higher order aggregates. At 2.0 and 2.5 mm MgCl2, a very long decay time of approximately 430 μs is observed (τ7in Table III), which is not detected in the case of vimentin. In Table IV, the weighted average values of the various relaxation times with their assignments are given. We do not use decay times with a relative contribution less than 10% in the calculation, since their precise value is difficult to estimate.Table IVAverage-relaxation times (τav) (μs) and their assignment in vimentin and GFAPτavvimentinτav GFAPAssignment4-asee Results and Discussion for assignments.μsμsτ1A1.3 ± 0.4Dimer rotation and flexibilityτ1B1.0 ± 0.3Flexibility of higher oligomerτ25.4 ± 1.05.7 ± 0.8Rotation staggered ∼65-nm building blockτ3A17 ± 4Particle with le" @default.
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• W2025972448 title "Transient Electric Birefringence Study of Intermediate Filament Formation from Vimentin and Glial Fibrillary Acidic Protein" @default.
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