Quantitative Analysis of Tropomyosin Linear Polymerization Equilibrium as a Function of Ionic Strength

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 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. 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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. 1998; PubMed Scopus Google the first residues in the fragment N.J. Hitchcock-DeGregori S.E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). structure of full-length Tm has to due to its high and 2000; PubMed Scopus Google Scholar). Tm show a repeat of that for a molecule containing residues was to to the between Tm W. J. Mol. Biol. 1969; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). of more C-terminal or modification of the group of Tm polymerization Smillie L.B. 16: PubMed Scopus Google Scholar). et al. A.S. Smillie L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar) show that native which is at has a viscosity actin and binding was the for both and Stewart A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google Scholar) propose a for the Tm head-to-tail interaction N-terminal and C-terminal residues from tropomyosin in which to hydrophobic and electrostatic interactions. of the of the tropomyosin head-to-tail interaction J. Biochem. Scopus (13) Google Scholar, K. Biochem. Biophys. 1962; PubMed Scopus Google Scholar, K. K. J. Biochem. 2000; PubMed Scopus (38) Google Scholar) due to the of containing of J. Biochem. Scopus (13) Google Scholar) to calculate as a function of ionic strength in the range mm 2 mm et al. K. Biochem. Biophys. 1962; PubMed Scopus Google Scholar) at and et al. K. K. J. Biochem. 2000; PubMed Scopus (38) Google Scholar) studies the head-to-tail interaction recombinant and C-terminal tropomyosin groups at between 2 and for ionic that from to which is in of the ionic strength dependence of viscosity of tropomyosin in this of the of the Tm head-to-tail interaction important for the interpretation of binding isotherms between Tm and other thin filament that Tm polymerization also its interactions with and Because Tm interactions with these proteins ionic a analysis of the ionic strength dependence of polymerization is also this in produced a recombinant tropomyosin with a 5-hydroxytryptophan at position 15 residues from the C terminus of the at other positions in this region specifically to TnT binding and actin binding A.D. C.S. Biophys. J. 2001; Scholar, A.D. C.S. Reinach F.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We show that the fluorescence of this is to Tm polymerization and this property to study polymerization as a function of ionic strength. at position 269 of the chicken skeletal α-tropomyosin Reinach F.C. Res. 1987; PubMed Scopus Google Scholar) was substituted for a tryptophan by the and (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 the of tropomyosin with a dipeptide Ala-Ser N-terminal fusion in of the N-terminal or non-fusion were the of or of these were and and as M. L. S. S.E. 1990; 6: PubMed Scopus Google Scholar). was or and in the of and was confirmed by S. Proc. Natl. Acad. Sci. U. S. A. 2001; Scopus Google Scholar). and of the containing 5-hydroxytryptophan at position 269 and were as C.S. Reinach F.C. PubMed Scopus Google Scholar). recombinant ASTm and were expressed and as (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 skeletal troponin troponin and TnT were expressed and and the troponin was as Ferro J.A. P.B. Reinach F.C. Sci. 2: PubMed Scopus Google Scholar, C.S. C.A. K. Smillie Reinach F.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Tropomyosin and troponin concentration were to the by Biochem. PubMed Scopus Google Scholar) ASTm as a = (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google was from chicken muscle J.D. J.A. PubMed Scopus Google and its concentration was by its at 1978; PubMed Scopus Google Scholar). the tropomyosin and in troponin were in fluorescence mm 50 mm mm and mm were and at for at binding was by of and to the and in Nature. PubMed Scopus Google Scholar). Recombinant were 50 mm mm mm and and circular dichroism studies were a at were at a with a time of in to an Tm in fluorescence at the concentration for at of containing with 5-hydroxytryptophan residues were a and of the ionic strength the of was in fluorescence with from to mm and Tm between and of in to the fluorescence intensity of a of the protein at a were for at the and the intensity was by the at between and fluorescence intensity was for the which was at high Tm and We with the that the in or with fluorescence intensity in see and concentration is to the polymer polymer has C terminus its length the ionic strength the affinity of the head-to-tail interaction Tm is in the at which the of the in to the fluorescence of the in were with to the Tm concentration and the for We as the of in the the of Tm C not in a head-to-tail and the and fluorescence both with to protein concentration and is also to is the average of polymer 2 is the tropomyosin is the and is the affinity constant for the head-to-tail interaction M. J. Mol. Biol. 1962; 4: PubMed Scopus Google Scholar, of W. and Scholar). We show the Because = at the as analysis of polymerization and M. J. Mol. Biol. 1962; 4: PubMed Scopus Google Scholar) show which and may to in of and and may to in of and as and were used to the in to for ionic the a = at ionic strength, the of which in for a or for were not and for the ionic in (between I = 0.045 and 0.095). by of at ionic or this range mm the in fluorescence as a function of tropomyosin concentration was to We a of recombinant Tm containing 5-hydroxytryptophan at a of positions its primary at proteins not We that the fluorescence of 5-hydroxytryptophan at position 269 is to ionic strength which to Tm Although the of the the fluorescence intensity as the concentration is from to mm fluorescence of the with tryptophan of at the position is also to ionic strength, in this the in intensity is by a not This intensity analysis in of a equilibrium Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar). this to the of the intensity of fluorescence of to the head-to-tail interaction of at fluorescence from the C.S. Reinach F.C. PubMed Scopus Google K. Reinach F.C. C.S. PubMed Scopus (13) Google Scholar) of Tm containing tryptophan of 5-hydroxytryptophan to the fluorescence of the non-fusion at position 269 that does not is to ionic strength the fluorescence of other Tm with 5-hydroxytryptophan at positions in the primary structure and A.D. C.S. Biophys. J. 2001; in the (position or in the N-terminal half of the protein and C.S. Reinach F.C. PubMed Scopus Google to ionic strength We to the at position both in the and of the dipeptide N-terminal other properties of Tm such as circular and viscosity in the of the proteins and were that the not the structure of the structure changes by the due to in the of this Because residue 269 to an external position in the heptad repeat of tropomyosin A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google the interactions at the interface between the in the coiled-coil structure not viscosity of the recombinant was at a of ionic (19Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google recombinant with an N-terminal Ala-Ser dipeptide fusion viscosity non-fusion recombinant viscosity at ionic We not between the of ASTm and or between and This that the at position 269 does not to changes in the interactions responsible for tropomyosin We the actin and troponin binding of the in and not that is able to bind actin and the binding of troponin to thin 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). does not bind actin its does bind in the of as for and 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). analysis that the at position 269 does not tropomyosin interactions with actin or troponin or its Because the between and is the or of an N-terminal dipeptide Ala-Ser fusion from the that the fluorescence intensity of the at position 269 is the head-to-tail interaction responsible for tropomyosin a for the polymerization of tropomyosin in which the 5-hydroxytryptophan in of a in the fluorescence the C-terminal of the tropomyosin molecule to which the is with the N-terminal of tropomyosin and a fluorescence which at the C-terminal of tropomyosin polymer and in the of in the is to the polymer concentration of length = of in is a function of the tropomyosin concentration and the equilibrium constant of the head-to-tail interaction the and M. J. Mol. Biol. 1962; 4: PubMed Scopus Google Scholar) show that that this equilibrium constant is of calculate the concentration of as a function of the and the Tm concentration We the of the tropomyosin head-to-tail interaction by the ionic strength dependence of the fluorescence intensity at a of Tm as for the ionic strength-dependent changes in fluorescence intensity of low ionic strength and high Tm the (for fluorescence intensity of the is at high ionic strength and low protein the fluorescence of the is This is also in the of polymerization at ionic was tropomyosin M. J. Mol. Biol. 1962; 4: PubMed Scopus Google the of polymerization at a ionic strength to the protein was to to the affinity for the head-to-tail interaction at a of ionic see that between = = and = = the equilibrium constant for the head-to-tail interaction by almost 2 orders of magnitude and ionic or this the in fluorescence as a function of tropomyosin concentration was to K. a of an from which at ionic strength may = This with and = describe the of and Tm concentration This is as a in see that the range I and may from to Because the between is not over a range of ionic and J. Chem. Scopus Google Scholar, S. PubMed Scopus Google Scholar, S.E. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google the in as this a for quantitatively Tm head-to-tail We a skeletal α-tropomyosin with a 5-hydroxytryptophan at position 269 fluorescence is or not the C terminus of the protein is in a head-to-tail interaction with Tm We at this from a of of as fluorescence with to is to Tm concentration in a as for a polymerization fluorescence is to ionic strength in a that the ionic strength dependence of Tm of the which is at the C-terminal of is the of an Ala-Ser dipeptide fusion at the N-terminal of the This dipeptide fusion is for the polymerization of recombinant skeletal Tm expressed in coli and P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). 5-hydroxytryptophan at position 269 is 15 residues from the C-terminal of the molecule of of that the head-to-tail interaction N-terminal residues and C-terminal residues from tropomyosin molecule A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google Scholar, Nature. 1979; PubMed Scopus Google Scholar). Because a tropomyosin with N-terminal may the in ASTm and to the first and residues of the in the the at position 269 is from the α-amino terminus of the tropomyosin by which this may the of the and its fluorescence the of the changes in the local coiled-coil and changes in is that the fluorescence of 5-hydroxytryptophan a up the Tm primary and to Tm polymerization A.D. C.S. Biophys. J. 2001; Scholar). This that changes in the coiled-coil structure by tropomyosin polymerization may to the and N-terminal of the a of the of at other positions in the molecule may to this view in the of the to the polymerization of the C-terminal to which is has to quantitatively the of Tm in head-to-tail in which the Tm concentration and ionic strength this to polymerization M. J. Mol. Biol. 1962; 4: PubMed Scopus Google Scholar, of W. and Scholar) to calculate the head-to-tail as a function of ionic strength as well as the average polymer length and polymer length is to the the tropomyosin structure dipeptide fusion and 5-hydroxytryptophan at position the of the head-to-tail interaction with the We these to the 5-hydroxytryptophan does not the of ASTm in viscosity and ASTm is from chicken muscle tropomyosin in actin and (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). a reports in the to which may J. Biochem. Scopus (13) Google of a for at ionic of results in to these an constant a with the that tropomyosin is these at and mm mm in et al. K. Biochem. Biophys. 1962; PubMed Scopus Google Scholar) of Tm to calculate a of at I = which is in with of at this ionic strength. et al. K. Biochem. 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Biochem. 2000; PubMed Scopus (38) Google Scholar) the polymerization properties of with or at position these full-length at low ionic strength, the by sedimentation equilibrium analysis of recombinant and C-terminal fragments were the at I = mm This is = at I = to calculate of the head-to-tail interaction over a range of ionic results an high to ionic for between I = 0.045 and a in the of by almost 2 orders of magnitude This is the first in which the constant for the tropomyosin head-to-tail interaction has at a of ionic a ionic strength dependence for interactions by electrostatic and is with the between and over a ionic strength range in This = to ionic strength that this with and = to describe the of and Tm concentration This is as a in This ionic strength and Tm concentration to average polymer the in with ionic changes in tropomyosin may in from the over a range of ionic the between is not J. Chem. Scopus Google Scholar, S. PubMed Scopus Google Scholar, S.E. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). between the of protein binding and ionic strength in of the to the at the interaction interface S.E. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). = is the constant at and the of the and C-terminal in the is the between charged and and = = at S.E. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. PubMed Scopus Google Scholar). from were to this to for and the over a range of between and is to the of the of the at the interface is and is to and is to a more of and 15 is to first and 15 residues of ASTm and tropomyosin head-to-tail is to residues from both the N and C of the tropomyosin A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google salt residues up to and the in the of may the Ala-Ser N-terminal fusion to this to residues of the first N-terminal residues has and 2 the α-amino the C-terminal has the C and residues may and the the N and C to salt between a positively charged N-terminal and a charged C-terminal of is more to in of at which and Stewart A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google Scholar) a for the tropomyosin head-to-tail residues from terminus that via the of the salt the of and This a of of of the tropomyosin and C-terminal coiled-coil the tropomyosin structures as N.J. Hitchcock-DeGregori S.E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, 2000; PubMed Scopus Google that in to the salt by and Stewart A.D. Stewart M. J. Mol. Biol. PubMed Scopus Google the positively charged the N terminus of its to a salt with the charged C-terminal of the also that the of residues and from and and from may a of and ionic interactions that in the of the head-to-tail of these ionic interactions with the of the N-terminal dipeptide the of to charged and positively charged and to interactions over a range of up to the of responsible for the ionic strength of Tm a of this the of the structure of the head-to-tail by high resolution or Tm to actin (for review, see 11Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar). produced by A.S. K. Smillie L.B. J. Biol. Chem. Full Text PDF PubMed Google modification Smillie L.B. 16: PubMed Scopus Google or recombinant (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) affinity for This at first that the that polymerization actin This is with the that binding Tm viscosity (18Hitchcock-DeGregori S.E. Heald R.W. J. Biol. Chem. 1987; 262: 9730-9735Abstract Full Text PDF PubMed Google Scholar, S. A. J. Biochem. 58: PubMed Scopus Google Scholar) and restores the actin binding of Tm (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, A.S. K. Smillie L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Smillie L.B. J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). and reports that the head-to-tail interaction between Tm more actin binding the of actin binding J. Hitchcock-DeGregori S.E. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, Butters C.A. L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Butters C.A. L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.A. L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.W. Hitchcock-DeGregori S.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. K. Hitchcock-DeGregori S.E. PubMed Scopus Google Scholar). et al. Butters C.A. L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) show that Tm binding to actin by the affinity of Tm for actin with in Tm polymerization were the of Tm an in in the of troponin. these due to the low the of Tm binding to actin in the of other proteins has not for with or not the head-to-tail to the of Although the head-to-tail is of for actin that the of the tropomyosin molecule also to of this affinity et al. J. Hitchcock-DeGregori S.E. 2000; PubMed Scopus Google Scholar) an affinity constant of recombinant skeletal of in that to at mm Our results show that at I = or 2 bind does not and not these and of to in the ASTm is able to bind actin in which is in the does not bind actin the Because to the N or C terminus of Tm Tm binding to that is the structure of the head-to-tail that actin binding and not the N-terminal or C-terminal of the the to which the head-to-tail actin stabilizes and its ionic interactions from by in the ionic strength of the modification that the structure of the N or C terminus to the structure of the actin binding affinity as recent high resolution structures of acetylated and of the N-terminal fragments of tropomyosin α-amino Tm polymerization and actin binding (22Greenfield N.J. Montelione G.T. Farid R.S. Hitchcock-DeGregori S.E. 1998; PubMed Scopus Google Scholar, N.J. Hitchcock-DeGregori S.E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). the of the dipeptide N-terminal repulsions between the positively charged α-amino groups at position a in the hydrophobic interface to destabilize the which may important for the of the head-to-tail responsible for this a tropomyosin fluorescence reports or not the C terminus of the molecule to which is in a head-to-tail interaction with the N terminus of tropomyosin We used this property to the affinity constant for this interaction at a of ionic and that the constant almost 2 orders of magnitude over a 50 mm in salt This ionic strength dependence the of a of charged residues in ionic interactions at the head-to-tail Our analysis to the average polymer length a of Because tropomyosin with and as this parameter may of in the analysis of Tm binding isotherms and in the interpretation of the and of thin filament and the by the binding of Ca2+ and We for with Alegria for with viscosity and Reinach for