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Revisado por pares
Quantitative Analysis of Tropomyosin Linear Polymerization Equilibrium as a Function of Ionic Strength
2002; Elsevier BV; Volume: 277; Issue: 3 Linguagem: Inglês
10.1074/jbc.m109568200
ISSN1083-351X
AutoresAurea D. Sousa, Chuck S. Farah,
Tópico(s)Viral Infections and Immunology Research
ResumoTropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken α-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin. Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken α-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin. tropomyosin troponin troponin T dithiothreitol recombinant skeletal muscle α-tropomyosin expressed in E. coli recombinant skeletal muscle α-tropomyosin expressed in E. coli with an Ala-Ser fusion at the N-terminal rfTm with Ala-269 substituted by 5-hydroxytryptophan ASTm with the Ala-269 substituted by tryptophan and 5-hydroxytryptophan, respectively electrostatic units circular dichroism 4-morpholinepropanesulfonic acid Skeletal muscle tropomyosin (Tm)1 is a 284-residue dimeric in-register coiled-coil protein that plays a central role in the regulation of muscle contraction through its interactions with actin and troponin (Tn) in the thin filament (1Hanson J. Lowy J. J. Mol. Biol. 1963; 6: 46-60Crossref Scopus (318) Google Scholar, 2Ebashi S. Endo M. Otsuki I. Q. Rev. Biophys. 1969; 2: 351-384Crossref PubMed Scopus (536) Google Scholar, 3Smillie L.B. Trends Biochem. Sci. 1979; 4: 151-155Abstract Full Text PDF Scopus (168) Google Scholar, 4Holmes K.C. Popp D. Gebhard W. Kabsh W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1325) Google Scholar, 5Tobacman L.S. Butters C.A. J. Mol. Biol. 2000; 275: 27587-27593Scopus (109) Google Scholar). Analysis of its primary structure revealed the existence of an extensive and almost perfect heptad repeat (abcdefg) in which hydrophobic residues at positions a and d create a dimerization interface that stabilizes the coiled-coil structure (6Sodek J. Hodges R.S. Smillie L.B. Jurasek L. Proc. Natl. Acad. Sci. U. S. A. 1962; 62: 3800-3804Google Scholar,7Stone D. Smillie L.B. J. Biol. Chem. 1978; 253: 1137-1148Abstract Full Text PDF PubMed Google Scholar). Further analysis of tropomyosin sequences also uncovered seven 39.5-residue pseudo-repeats, which may reflect the tropomyosin 7:1 binding stoichiometry with actin (8McLachlan A.D. Stewart M. J. Mol. Biol. 1976; 103: 271-298Crossref PubMed Scopus (300) Google Scholar). Also associated with both tropomyosin and actin is the troponin complex, which consists of the Ca2+ binding subunit (troponin C), the inhibitory subunit (troponin I), and the tropomyosin binding subunit (TnT). Tn-Tm interactions occur mainly between the C-terminal half of TnT and the region near position 190 of Tm and between the N-terminal half of TnT and the C-terminal of the Tm molecule (for review, see Refs.9Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (447) Google Scholar, 10Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 11Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar, 12Squire J.M. Morris E.P. FASEB J. 1998; 12: 761-771Crossref PubMed Scopus (168) Google Scholar, 13Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1342) Google Scholar). Muscle tropomyosin high viscosity at low ionic strength was noted from the time of its first isolation (14Bailey K. Biochem. J. 1948; 43: 271-279Crossref PubMed Scopus (345) Google Scholar). Soon thereafter Tsao et al. (15Tsao T.-C. Bailey K. Adair G.S. Biochem. J. 1951; 49: 27-35Crossref PubMed Scopus (38) Google Scholar) describe the ionic strength dependence of the viscosity in more detail and Kay and Bailey (16Kay C.M. Bailey K. Biochim. Biophys. Acta. 1960; 40: 149-156Crossref PubMed Scopus (26) Google Scholar) relate changes in the polymer length with salt concentration and propose that polymerization is due to simple end-to-end or head-to-tail aggregation, which was later confirmed by in-depth studies of cardiac Tm via sedimentation velocity, sedimentation equilibrium, osmometry, viscometry, and optical rotatory dispersion (17McCubbin W.D. Kay C.M. Can. J. Biochem. 1969; 47: 411-414Crossref PubMed Scopus (13) Google Scholar). Although muscle Tm is acetylated at its N terminus, recombinant non-fusion Tm (nfTm) expressed in bacteria lacks this modification and does not polymerize or bind to actin (18Hitchcock-DeGregori S.E. Heald R.W. J. Biol. Chem. 1987; 262: 9730-9735Abstract Full Text PDF PubMed Google Scholar, 19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). Recombinant Tm expressed in insect cells (20Urbancikova M. Hitchcock-DeGregori S.E. J. Biol. Chem. 1994; 269: 14310-14315Abstract Full Text PDF Google Scholar) or yeast (21Hilario E. Lataro R.C. Alegria M.C. Lavarda S.C. Ferro J.A. Bertolini M.C. Biochem. Biophys. Res. Commun. 2001; 284: 955-960Crossref PubMed Scopus (8) Google Scholar) has its N terminus acetylated and is functional. Because the N-acetylated initiation methionine in the native protein occupies an internal position in the coiled-coil structure (position a), repulsions between the positively charged α-amino groups in the recombinant protein were thought to destabilize the local coiled-coil structure as well as the head-to-tail interaction responsible for polymerization (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). An N-terminal dipeptide fusion (ASTm) reduces these repulsions by placing the charged α-amino group at an external position (f) and restores the polymerization and actin binding properties of Tm (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). This view is confirmed by the recent high resolution structures of N-terminal fragments of acetylated and non-acetylated tropomyosin; the acetylated fragment maintains the coiled-coil structure up to the N terminus (22Greenfield N.J. Montelione G.T. Farid R.S. Hitchcock-DeGregori S.E. Biochemistry. 1998; 37: 7834-7843Crossref PubMed Scopus (81) Google Scholar), whereas the first two residues in the unacetylated fragment are non-helical (23Brown J.H. Kim K.H. Jun G. Greenfield N.J. Dominguez R. Volkmann N. Hitchcock-DeGregori S.E. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8496-8501Crossref PubMed Scopus (218) Google Scholar). The structure of full-length Tm has only been resolved to 7 Å due to its high flexibility and asymmetry (24Whitby F.G. Phillips Jr., G.N. Proteins. 2000; 38: 49-59Crossref PubMed Scopus (158) Google Scholar). Tm crystals show a repeat distance of 410 Å, less than that expected for a molecule containing 284 α-helical residues (∼423 Å). The size difference was proposed to correspond to the overlap between Tm molecules (25Caspar D.L. Cohen C. Longley W. J. Mol. Biol. 1969; 41: 87-107Crossref PubMed Scopus (231) Google Scholar, 26Cohen C. Caspar D.L. Parry D.A. Lucas R.M. Cold Spring Harbor Symp. Quant. Biol. 1972; 36: 205-216Crossref PubMed Scopus (66) Google Scholar). The removal of more than three C-terminal amino acids or chemical modification of the ε-NH2 group of Lys-7 abolishes Tm polymerization (27Johnson P. Smillie L.B. Biochemistry. 1977; 16: 2264-2269Crossref PubMed Scopus (90) Google Scholar). Heeley et al. (28Heeley D.H. Watson W.H. Mak A.S. Dubord P. Smillie L.B. J. Biol. Chem. 1989; 264: 2424-2430Abstract Full Text PDF PubMed Google Scholar) show that native Tm, which is phosphorylated at Ser-283, has a greater viscosity than unphosphorylated Tm, but actin and Tn binding ability was the same for both forms. McLachlan and Stewart (29McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (571) Google Scholar) propose a model for the Tm head-to-tail interaction involving nine N-terminal and C-terminal residues from each tropomyosin chain, in which they attempted to maximize hydrophobic and electrostatic interactions. Very few quantitative measures of the thermodynamics of the tropomyosin head-to-tail interaction have been performed (30Asai H. J. Biochem. (Tokyo). 1961; 50: 183-189Crossref Scopus (13) Google Scholar, 31Ooi T. Mihashi K. Kobayashi H. Arch. Biochem. Biophys. 1962; 98: 1-11Crossref PubMed Scopus (40) Google Scholar, 32Sano K. Maeda K. Oda T. Maeda Y. J. Biochem. (Tokyo). 2000; 127: 1095-1102Crossref PubMed Scopus (38) Google Scholar) due to the heterogeneity of solutions containing polymers of different lengths. Asai (30Asai H. J. Biochem. (Tokyo). 1961; 50: 183-189Crossref Scopus (13) Google Scholar) attempts to calculate K as a function of ionic strength but only in the range 0–12 mm KCl, 2 mm phosphate, whereas Ooi et al. (31Ooi T. Mihashi K. Kobayashi H. Arch. Biochem. Biophys. 1962; 98: 1-11Crossref PubMed Scopus (40) Google Scholar) calculatesK at 0.1 and 1 m. Sano et al. (32Sano K. Maeda K. Oda T. Maeda Y. J. Biochem. (Tokyo). 2000; 127: 1095-1102Crossref PubMed Scopus (38) Google Scholar) studies the head-to-tail interaction using recombinant N- and C-terminal tropomyosin fragments. All groups arrived at Kvalues between 2 × 104 and 1.25 × 105m−1 for ionic strengths that varied from less than 0.01 to 1.0, which is surprising in light of the strong ionic strength dependence of viscosity of micromolar tropomyosin solutions in this range. Quantitative measures of the thermodynamics of the Tm head-to-tail interaction are important for the interpretation of binding isotherms between Tm and other thin filament proteins, especially since mutations that influence Tm polymerization also affect its interactions with Tn and actin. Because Tm interactions with these proteins are strongly ionic strength-dependent, a quantitative analysis of the ionic strength dependence of polymerization thermodynamics is also important. With this aim in mind, we have produced a recombinant tropomyosin with a fluorescent 5-hydroxytryptophan probe at position 269, located 15 residues from the C terminus of the polypeptide chain. 5-Hydroxytryptophan probes at other positions in this region are specifically sensitive to TnT binding and actin binding (33Sousa A.D. Farah C.S. Biophys. J. 2001; 80 (Abstr. 407–POS): 91Google Scholar, 34Oliveira D.M. Nakaie C.R. Sousa A.D. Farah C.S. Reinach F.C. J. Biol. Chem. 2000; 275: 27513-27519Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We show that the fluorescence of this probe is sensitive to Tm polymerization and use this property to study polymerization thermodynamics as a function of ionic strength. The alanine codon at position 269 of the chicken skeletal α-tropomyosin cDNA (35Gooding C. Reinach F.C. Macleod A.R. Nucleic Acids Res. 1987; 158105Crossref PubMed Scopus (18) Google Scholar) was substituted for a tryptophan codon by PCR-mediated site-directed mutagenesis using the bacterial expression vector constructs pET-MASTmy and pET-Tmy (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar) as templates. These vectors direct the expression of tropomyosin with a dipeptide Ala-Ser N-terminal fusion (after in vivo removal of the N-terminal methionine) or non-fusion tropomyosin, respectively. The PCR reactions were performed using the following pairs of oligonucleotides: oligoBSTEII+, 5′-AAGAGATCCAGCTTAAAGAAG-3′, plus oligoA269W−, 5′-CCTCGCTGATCCATTTGTACTTC-3′, or oligo EcoRI−, 5′-CATTAACCTATAAAAATAGGCG-3′, plus oligoA269W+, 5′-GAAGTACAAATGGATCAGCGAGG-3. The products of these two reactions were combined, denatured, renatured, and amplified using oligoBstEII+ and oligo EcoRI− as described (36Higuchi M. Wong C. Kochhan L. Aronis S. Kasper C.K. Kazazian Jr., H.H. Antonarakis S.E. Genomics. 1990; 6: 65-71Crossref PubMed Scopus (69) Google Scholar). The amplified product was subcloned into pET-MASTmy or pET-Tmy previously digested withBstEII and EcoRI, resulting in the production of pET-MAS269W and pET-nf269W. The nucleotide sequence was confirmed by dideoxy sequencing (37Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 74: 5463-5467Crossref Scopus (52770) Google Scholar). Expression and purification of the mutant tropomyosins containing 5-hydroxytryptophan at position 269 (AS269(5OHW) and nf269(5OHW)) were performed as described previously (38Farah C.S. Reinach F.C. Biochemistry. 1999; 38: 10543-10551Crossref PubMed Scopus (23) Google Scholar). The recombinant tropomyosins ASTm and nfTm were expressed and purified as described (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar) Recombinant chicken fast skeletal troponin I, troponin C, and TnT were expressed and purified separately, and the troponin complex was reconstituted as described (39Quaggio R.B. Ferro J.A. Monteiro P.B. Reinach F.C. Protein Sci. 1993; 2: 1053-1056Crossref PubMed Scopus (24) Google Scholar, 40Farah C.S. Myiamoto C.A. Ramos C.H. da Silva A.C. Quaggio R.B. Fujimori K. Smillie R.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Tropomyosin and troponin concentration were determined according to the method described by Hartree (41Hartree E.F. Anal. Biochem. 1972; 48: 422-427Crossref PubMed Scopus (4558) Google Scholar) using ASTm as a standard (ε 278nm1% = 2.9 (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar)). Actin was prepared from chicken pectoral muscle (42Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-182Crossref PubMed Scopus (984) Google Scholar), and its concentration was determined by its absorbance at 290 nm (43Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar). Actin (14 μm), the specified tropomyosin (2 μm) and in some cases troponin (2 μm) were combined in fluorescence buffer (25 mm MOPS, pH 7.0, 50 mm NaCl, 5 mm MgCl2, and 1 mm DTT). The mixtures were homogenized and centrifuged at 315,000 × g for 20 min at 25 °C. In qualitative assays, binding was detected by analyzing equivalent volumes of mixtures before centrifugation and samples corresponding to the supernatant and pellet after centrifugation in 15% SDS-PAGE (44Laemmili U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Recombinant Tms were dialyzed against 50 mm NaH2PO4, pH 7.0, 0.5 mm DTT, 0.5 mm EDTA, and 100 mmKCl, and circular dichroism studies were carried out using a Jasco 720 spectropolarimeter at 25 °C. Viscosity measurements were carried out at 21 °C using a Cannon-Manning semi-microviscometer (A50) with a buffer outflow time of 236–241 s. Each point in Fig.2A corresponds to an 8-μm Tm solution in fluorescence buffer at the specified NaCl concentration after equilibration for 7 h at 21 ± 1 °C. Fluorescence spectra of mixtures containing tropomyosins with 5-hydroxytryptophan residues were determined using a F-4500 Hitachi spectrofluorimeter (excitation, 312 nm; excitation and emission slit bandwidths, 5 nm; temperature, 25 °C). The effect of the ionic strength on the emission spectrum of AS269(5OHW) was determined in fluorescence buffer with NaCl concentrations varying from 10 to 350 mm and Tm concentrations varying between 0.5 and 8 μm. Each point of each curve in Fig. 3A corresponds to the fluorescence intensity of a 1.5-ml mixture of the protein at a specific NaCl concentration. Samples were equilibrated for 7 h at 25 °C before taking the spectra, and the emission intensity was determined by summing the emissions at all wavelengths between 330 and 380 nm. The fluorescence intensity was corrected for the inner filter effect, which was only significant at high Tm concentrations and never exceeded 14%. We begin with the premise that the probe can exist in two environments, free or "polymerized," with different fluorescence intensity in each state (Fmin andFmax, respectively; see Figs. 1 and 3). The free probe concentration is equal to the polymer concentration, since each polymer has only one C terminus regardless its length (Fig.1D). As the ionic strength increases, the affinity of the head-to-tail interaction decreases until essentially all Tm is in the monomeric state, at which point the base line of the titration (see thearrow in Fig. 3A) corresponds to the intrinsic fluorescence of the free probes (Fmin). The data in Fig. 3A were normalized with respect to the Tm concentration and the Fmin for each curve. We define fo as the fraction of probes in the free environment, i.e. the fraction of Tm molecules whose C termini are not involved in a head-to-tail interaction.fo=(FmaxN−FN)/(FmaxN−1)Equation 1 where FN and FmaxNare, respectively, the actual and maximal fluorescence intensities, both normalized with respect to protein concentration and toFmin. fo is also equivalent to −1, where is the average number of monomers per polymer chain,fo=〈i〉−1=[polymers]/[λtot]Equation 2 =(λ1+λ2+λ3…λi)/λtot=∑i=1Ki−1λ1i/λtotwhere λtot is the total tropomyosin concentration, λi is the i-mer concentration, and K is the affinity constant for the head-to-tail interaction (45Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (564) Google Scholar, 46Cantor C.R. Schimmel P.R. Biophysical Chemistry. Part I: The Conformation of Biological Macromolecules. W. H. Freeman and Co., New York1980: 145-149Google Scholar). We can then show the following.fo=∑i=1K−1·Kiλ1i/λtot=∑i=1(Kλ1)i/KλtotEquation 3 Because Σxi = x/(1−x), we arrive at the expression,fo=λ1/[λtot(1−Kλ1)]Equation 4 so long as λ1 < K−1. In their analysis of linear polymerization equilibria, Oosawa and Kasai (45Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (564) Google Scholar) show that,λtot=∑iλi=λ1/(1−Kλ1)2Equation 5 which can be rearranged toλ1=(2Kλtot+1−(4Kλtot+1)1/2)/(2K2λtot)Equation 6 Equations 4 and 6 may be combined to expressfo in terms of K and λtotas follows.fo=(2/[(4Kλtot+1)1/2−1])−(1/Kλtot)Equation 7 Finally, Equations 1 and 7 may be combined to expressFN in terms of FmaxN,K, and λtot as follows.FN=FmaxN−((FmaxN−1)[(2/[(4Kλtot+1)1/2−1])−(1/Kλtot)])Equation 8 Equations 7 and 8 were used to fit the experimental data in Fig.3B to simultaneously obtain values for K andFmaxN (at each ionic strength) using the program SigmaPlot 3.0 (Jandel Scientific). Using a fixed value ofFmaxN = 1.55 (see Fig. 3A), we recalculated K at each ionic strength, the values of which are shown in Fig. 4. The values for K obtained using a fixed or variable value for FmaxN were not significantly different (±50% and usually much less) for the ionic strengths shown in Fig. 4 (between I = 0.045 and 0.095). As can be seen by inspection of Fig. 3, at ionic strengths below or above this range (20–70 mm NaCl), the observed change in intrinsic fluorescence as a function of tropomyosin concentration was too little to accurately estimateK. We analyzed a series of recombinant Tm mutants containing 5-hydroxytryptophan at a number of positions along its primary structure. The fluorescent probe can be selectively excited at wavelengths above 300 nm, where tryptophan-containing proteins do not absorb (Fig. 1C). We found that the fluorescence of 5-hydroxytryptophan incorporated at position 269 (AS269(5OHW)) is sensitive to ionic strength variations, which lead to Tm depolymerization (Fig. 1A). Although the λmax of the emission spectrum remains constant, the fluorescence intensity decreases ∼38% as the NaCl concentration is raised from 10 to 300 mm (Fig. 1A). The fluorescence of the mutant with tryptophan (instead of 5-hydroxytryptophan), incorporated at the same position (AS269W), is also sensitive to ionic strength, but in this case the decrease in intensity is accompanied by a red shift (data not shown). This combined spectral shift plus intensity change complicates quantitative analysis in terms of a two-state equilibrium (47Eftink M.R. Biophys. J. 1994; 66: 482-501Abstract Full Text PDF PubMed Scopus (451) Google Scholar). For this reason, we chose to use the variation of the intensity of fluorescence of AS269(5OHW) to investigate the head-to-tail interaction of Tm. By exciting at 312 nm, we eliminate any fluorescence contribution from the small fraction (38Farah C.S. Reinach F.C. Biochemistry. 1999; 38: 10543-10551Crossref PubMed Scopus (23) Google Scholar,48Das K. Ashby K.D. Smirnov A.V. Reinach F.C. Petrich J.W. Farah C.S. Photochem. Photobiol. 1999; 70: 719-730Crossref PubMed Scopus (13) Google Scholar) of Tm molecules containing tryptophan instead of 5-hydroxytryptophan (Fig. 1C). In contrast to AS269(5OHW), the fluorescence of the non-fusion mutant labeled at position 269 that does not polymerize, nf269(5OHW), is insensitive to ionic strength variations (Fig. 1B). Furthermore, the fluorescence intensities of other polymerizable Tm mutants labeled with 5-hydroxytryptophan at nearby positions in the primary structure (positions 261, 263, and 267 (33Sousa A.D. Farah C.S. Biophys. J. 2001; 80 (Abstr. 407–POS): 91Google Scholar)), in the center (position 185), or in the N-terminal half of the protein (positions 90, 101, 111, and 122 (38Farah C.S. Reinach F.C. Biochemistry. 1999; 38: 10543-10551Crossref PubMed Scopus (23) Google Scholar)) are insensitive to ionic strength variations. We attempted to determine whether the mutation at position 269, both in the presence and absence of the dipeptide N-terminal fusion, significantly affected other properties of Tm such as actin-binding (+/−Tn), circular dichroism, and viscosity (Fig.2). No significant differences in the CD spectra of the four proteins (ASTm, nfTm, AS269(5OHW), and nf269(5OHW) were observed (Fig. 2C), indicating that the mutations do not significantly affect the overall secondary structure of the molecule. Local secondary structure changes induced by the mutation, however, cannot be ruled out due to limitations in the sensitivity of this method. Because residue 269 corresponds to an external position (c) in the heptad repeat of tropomyosin (29McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (571) Google Scholar), the stereospecific interactions at the interface between the two α-helices in the coiled-coil structure should not be disrupted. The kinematic viscosity of the four recombinant tropomyosins was determined at a series of ionic strengths (Fig.2A). As expected (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar), recombinant tropomyosins with an N-terminal Ala-Ser dipeptide fusion present salt-dependent viscosity dependence, whereas non-fusion recombinant tropomyosins have significantly reduced viscosity at all ionic strengths tested. We did not observe any significant difference between the viscosities of ASTm and AS269(5OHW) or between nfTm and nf269(5OHW) (Fig.2A). This indicates that the mutation at position 269 does not lead to significant changes in the intermolecular interactions responsible for tropomyosin polymerization. We checked the actin and troponin binding ability of the four tropomyosins in co-sedimentation assays (Fig. 2B and data not shown). These assays showed that AS269(5OHW) is able to bind actin and mediates the binding of troponin to thin filaments, similar to ASTm (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). Furthermore, nf269(5OHW) does not bind actin on its own but does bind in the presence of troponin, just as previously observed for nfTm (Fig. 2B and Ref. 19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). The above analysis indicates that the probe at position 269 does not significantly affect tropomyosin interactions with actin or troponin or its salt-dependent polymerization. Because the only chemical difference between AS269(5OHW) and nf269(5OHW) is the presence or absence of an N-terminal dipeptide Ala-Ser fusion ∼40 nm from the 5-hydroxtryptophan probe, we conclude that the fluorescence intensity of the probe at position 269 (Fig. 1A) is sensitive the head-to-tail interaction responsible for tropomyosin polymerization. Fig. 1D presents a scheme for the linear polymerization of tropomyosin in which the 5-hydroxytryptophan probe can exist in one of two environments, (i) a "polymerized" environment (filled circles in the figure, maximum fluorescence intensity), where the C-terminal end of the tropomyosin molecule to which the probe is covalently attached interacts with the N-terminal end of another tropomyosin molecule, and (ii) a free environment (open circles, minimum fluorescence intensity), which occurs only at the C-terminal ends of each tropomyosin polymer and in free monomers. Therefore, the fraction of probes in the free environment is directly proportional to the total polymer concentration (where monomers are considered polymers of chain length (i) = 1). The fraction of probes in each environment is a function of the total tropomyosin concentration and the equilibrium association constant (K) of the head-to-tail interaction under the conditions specified. Oosawa and Kasai (45Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (564) Google Scholar) show that if we assume that this equilibrium constant is independent of chain length, we can calculate the concentration of each species as a function of the K and the total Tm concentration (see "Experimental Procedures"). We therefore studied the thermodynamics of the tropomyosin head-to-tail interaction by monitoring the ionic strength dependence of the fluorescence intensity at a number of Tm concentrations, as described under "Experimental Procedures." Fig.3A shows representative curves for the ionic strength-dependent changes in fluorescence intensity of AS269(5OHW). At low ionic strength and high Tm concentrations, the normalized (for concentration) fluorescence intensity of the probe is maximum, whereas at high ionic strength and low protein concentrations the fluorescence of the probe is significantly reduced. This is also evident in Fig. 3B, where the extent of polymerization (1 − fo) at selected ionic strengths was plotted against tropomyosin concentration. As expected (45Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (564) Google Scholar), the extent of polymerization at a specific ionic strength can be seen to be dependent on the total protein concentration. The data was fit to Equation 8 to obtain the affinity constants for the head-to-tail interaction at a number of ionic strengths (Fig. 4). In Fig. 4B we see that between [NaCl] = 20 mm(I = 0.045) and [NaCl] = 70 mm(I = 0.095), the equilibrium constant for the head-to-tail interaction varies by almost 2 orders of magnitude (1.9 × 107m−1 ± 1.1 × 106 and 3.5 × 105m−1 ± 6.5 × 104, respectively). At ionic strengths below or above this range, the observed change in intrinsic fluorescence as a function of tropomyosin concentration was too little to accurately estimate K. However, a plot of log K versus[I]0.5 reveals an essentially linear relationship (Fig. 4B) from which K at any ionic strength may be interpolated (log K = 11.1 − 18.0[I]0.5). This result can be combined with Equation 7 and fo = −1to explicitly describe the interdependence of ,K, and total Tm concentration (λtot). This relationship is presented as a three-dimensional surface in Fig.5A. In Fig. 5B, we see that within the range 0.05 < I < 0.1 and 1 μm < [Tm] < 10 μm, may vary from 1 to 10. Because the relationship between log Kand I0.5 is certainly not truly linear over a wide range of ionic strengths (see Discussion and Refs. 49Pitzer K.S. Mayorga G. J. Physical Chem. 1973; 77: 2300-2308Crossref Scopus (1708) Google Scholar, 50Highsmith S. Biochemistry. 1997; 36: 2010-2016Crossref PubMed Scopus (7) Google Scholar, 51Song X.Z. Pedersen S.E. Biophys. J. 2000; 78: 1324-1334Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), the values shown in Fig. 5A withI < 0.05 should be considered only as rough estimates. In this work we have developed a new approach for quantitatively studying
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