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• W2002802503 abstract "Tropomodulin 1 (Tmod1) is a ∼40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd ∼ 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd ∼ 0.1 μm). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160–359) contains the principal actin-capping activity (Kd ∼ 0.4 μm), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1–130) contains a second, weaker actin-capping activity (Kd ∼ 1.8 μm). Interestingly, 160–359 but not 1–130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity. Tropomodulin 1 (Tmod1) is a ∼40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd ∼ 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd ∼ 0.1 μm). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160–359) contains the principal actin-capping activity (Kd ∼ 0.4 μm), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1–130) contains a second, weaker actin-capping activity (Kd ∼ 1.8 μm). Interestingly, 160–359 but not 1–130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity. Tropomodulins (Tmods) 1The abbreviations used are: Tmod, tropomodulin; E, N, U, and Sk-Tmod, erythrocyte, neural, ubiquitous, skeletal tropomodulin, respectively; TM, tropomyosin; mAb9, monoclonal antibody 9; GST, glutathione S-transferase; DTT, dithiothreitol; LRR, leucine-rich repeat; MES, 4-morpholineethanesulfonic acid. are a conserved family of actin filament pointed end-capping proteins that are present in vertebrates, flies, and worms. In vertebrates, there are four canonical ∼40-kDa isoforms: E, N, U, and Sk-Tmod (Tmod1 to -4, respectively), which are about 60–70% identical to one another and are expressed in a tissue-specific and developmentally regulated fashion (1Almenar-Queralt A. Lee A. Conley C.A. Ribas de Pouplana L. Fowler V.M. J. Biol. Chem. 1999; 274: 28466-28475Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 2Cox P.R. Zoghbi H.Y. Genomics. 2000; 63: 97-107Crossref PubMed Scopus (71) Google Scholar, 3Conley C.A. Fritz-Six K.L. Almenar-Queralt A. Fowler V.M. Genomics. 2001; 73: 127-139Crossref PubMed Scopus (105) Google Scholar). Tmod function in vivo is best understood in striated muscle cells, where Tmod1 or Tmod4 is associated with the free, pointed ends of thin filaments in sarcomeres and functions to regulate pointed end dynamics and control filament length (1Almenar-Queralt A. Lee A. Conley C.A. Ribas de Pouplana L. Fowler V.M. J. Biol. Chem. 1999; 274: 28466-28475Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 4Fowler V.M. Sussman M.A. Miller P.G. Flucher B.E. Daniels M.P. J. Cell Biol. 1993; 120: 411-420Crossref PubMed Scopus (133) Google Scholar, 5Gregorio C.C. Weber A. Bondad M. Pennise C.R. Fowler V.M. Nature. 1995; 377: 83-86Crossref PubMed Scopus (160) Google Scholar, 6Littlefield R. Almenar-Queralt A. Fowler V.M. Nat. Cell Biol. 2001; 3: 544-551Crossref PubMed Scopus (183) Google Scholar, 7Mardahl-Dumesnil M. Fowler V.M. J. Cell Biol. 2001; 155: 1043-1053Crossref PubMed Scopus (61) Google Scholar). Whereas the functions of Tmods in non-muscle cells are less well understood, recent work from our laboratory indicates that the Tmod3 isoform is present in the leading lamellipodia in human microvascular endothelial cells, where it reduces pointed end disassembly and negatively regulates cell migration (8Fischer R.S. Fritz-Six K.L. Fowler V.M. J. Cell Biol. 2003; 161: 371-380Crossref PubMed Scopus (84) Google Scholar). In the brain, the Tmod2 isoform may play a role in synaptic plasticity, based on characterization of behavioral deficits and enhanced long term potentiation in a Tmod2 knockout mouse (9Cox P.R. Fowler V.M. Xu B. Sweatt J.D. Paylor R. Zoghbi H.Y. Mol. Cell. Neurosci. 2003; 23: 1-12Crossref PubMed Scopus (60) Google Scholar). Tmods are unique in several respects as compared with all other capping proteins. First, Tmods bind specifically to actin filament pointed ends but not to actin monomers, sides of filaments, or barbed filament ends (10Fowler V.M. J. Cell Biol. 1990; 111: 471-482Crossref PubMed Scopus (131) Google Scholar, 11Weber A. Pennise C.R. Babcock G.G. Fowler V.M. J. Cell Biol. 1994; 127: 1627-1635Crossref PubMed Scopus (243) Google Scholar). Second, Tmods cap pointed ends transiently, thus slowing down monomer addition and leading to an increased proportion of filaments with terminal ADP-actin subunits that have a lower affinity for ends (12Weber A. Pennise C.R. Fowler V.M. J. Biol. Chem. 1999; 274: 34637-34645Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The consequence is that at high Tmod concentrations, there is net actin depolymerization and filament shortening until a new steady state is achieved. Third, Tmods are unique among all capping proteins in that they also bind to tropomyosins (TM) (10Fowler V.M. J. Cell Biol. 1990; 111: 471-482Crossref PubMed Scopus (131) Google Scholar, 13Sussman M.A. Fowler V.M. Eur. J. Biochem. 1992; 205: 355-362Crossref PubMed Scopus (28) Google Scholar, 14Watakabe A. Kobayashi R. Helfman D.M. J. Cell Sci. 1996; 109: 2299-2310Crossref PubMed Google Scholar) and their capping activity is enhanced several thousand-fold in the presence of TM (1Almenar-Queralt A. Lee A. Conley C.A. Ribas de Pouplana L. Fowler V.M. J. Biol. Chem. 1999; 274: 28466-28475Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11Weber A. Pennise C.R. Babcock G.G. Fowler V.M. J. Cell Biol. 1994; 127: 1627-1635Crossref PubMed Scopus (243) Google Scholar, 12Weber A. Pennise C.R. Fowler V.M. J. Biol. Chem. 1999; 274: 34637-34645Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 2Tmods can bind to both muscle and non-muscle TMs, but for simplicity in this study, “TM” is used to refer to skeletal muscle tropomyosin unless otherwise indicated. However, Tmod capping of TM-actin filament pointed ends does not affect the pointed end critical concentration, because the tightly capped TM-actin ends do not exchange actin subunits and thus are silent. We have proposed that TM enhances the actin-capping activity of Tmod by providing a second binding site for Tmod at the filament pointed end. However, it is also possible that TM binding to Tmod leads to a conformational change in the Tmod that increases its affinity for actin at the pointed ends. The role of TM binding for Tmod function in vivo is not known. Recent biophysical and biochemical studies indicate that the amino-terminal region of Tmod1 is highly protease-sensitive and unstructured, whereas the carboxyl-terminal portion is compact, folded, and protease-resistant (15Kostyukova A. Maeda K. Yamauchi E. Krieger I. Maeda Y. Eur. J. Biochem. 2000; 267: 6470-6475Crossref PubMed Scopus (53) Google Scholar, 16Kostyukova A. Tiktopulo E.I. Maeda Y. Biophys. J. 2001; 81: 345-351Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 17Fujisawa T. Kostyukova A. Maeda Y. FEBS Lett. 2001; 498: 67-71Crossref PubMed Scopus (34) Google Scholar, 18Greenfield N.J. Fowler V.M. Biophys. J. 2002; 82: 2580-2591Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The carboxyl-terminal portion (residues 160–344) of chicken Tmod1 (residues 1–359) has been crystallized, and its structure has been solved to a resolution of 1.45 Å (19Krieger I. Kostyukova A. Yamashita A. Nitanai Y. Maeda Y. Biophys. J. 2002; 83: 2716-2725Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This reveals that the carboxyl-terminal domain is composed of a series of five leucine-rich repeats, each consisting of an α-helix/β-sheet pair. The five leucine-rich repeats are followed by a nonhomologous, associated α-helix (α6) composed of residues 322–344. The structure of the final 15 amino acids (345–359) at the carboxyl terminus of Tmod1 is not known, since they were not present in the recombinant protein used for crystallization by Krieger et al. (19Krieger I. Kostyukova A. Yamashita A. Nitanai Y. Maeda Y. Biophys. J. 2002; 83: 2716-2725Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The TM-binding region of Tmod1 and Tmod4 is located in the amino-terminal portion between residues 1 and 130 (15Kostyukova A. Maeda K. Yamauchi E. Krieger I. Maeda Y. Eur. J. Biochem. 2000; 267: 6470-6475Crossref PubMed Scopus (53) Google Scholar, 18Greenfield N.J. Fowler V.M. Biophys. J. 2002; 82: 2580-2591Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Babcock G.G. Fowler V.M. J. Biol. Chem. 1994; 269: 27510-27518Abstract Full Text PDF PubMed Google Scholar, 21Sung L.A. Fowler V.M. Lambert K. Sussman M.A. Karr D. Chien S. J. Biol. Chem. 1992; 267: 2616-2621Abstract Full Text PDF PubMed Google Scholar). It was proposed some time ago that the carboxyl-terminal half of Tmod1 contained the actin pointed end-capping domain, based on the ability of a monoclonal antibody (mAb9) that bound to this region of Tmod1 to inhibit pointed end-capping activity (5Gregorio C.C. Weber A. Bondad M. Pennise C.R. Fowler V.M. Nature. 1995; 377: 83-86Crossref PubMed Scopus (160) Google Scholar). In this study, we have identified directly the actin pointed end-capping regions on Tmod1 by designing a series of recombinant fragments based on the domain structure of Tmod1 and testing them for their ability to inhibit actin elongation from the pointed ends of gelsolin-capped actin filaments in pyrene-actin polymerization assays. These experiments demonstrate that a TM-independent pointed end-capping activity for pure actin filaments is associated with the extreme carboxyl-terminal end of Tmod1, whereas a TM-regulated actin-capping activity is located in the unstructured TM-binding region in the amino-terminal portion of Tmod1. Unexpectedly, we also report that the full-length Tmod1 protein as well as the carboxyl-terminal domain enhances spontaneous actin nucleation, whereas the amino-terminal domain does not. These results suggest that 1) Tmod1 has two actin binding domains that interact with two different sites on actin filament pointed ends and 2) TM-binding by Tmod1 most likely enhances Tmod1 capping by enhancing the actin capping affinity of the Tmod1 amino-terminal actin-capping domain rather than by providing a separate TM-binding site for Tmod1 at the filament end. These biochemical results will provide a foundation for future exploration of the role of TM in regulation of actin-capping activities by Tmod1 in vivo. Construction of cDNAs and Expression of Recombinant Tmod1 Fragments—Full-length chicken Tmod1 (E-Tmod) (accession number L36678) was inserted in frame in the pGEX-KG vector so as to code for a fusion protein with glutathione S-transferase (GST) on the amino-terminal end of Tmod1 (20Babcock G.G. Fowler V.M. J. Biol. Chem. 1994; 269: 27510-27518Abstract Full Text PDF PubMed Google Scholar). cDNAs encoding GST fusion proteins with various Tmod1 fragments were inserted in the EcoRI and/or NcoI sites in the linker region of the pGEX-KG vector using the polymerase chain reaction with appropriate primers. The use of different insertion sites in the linker region for the truncated Tmod1 cDNAs resulted in some amino acid differences in the sequence of the linker remaining on the amino-terminal end of the purified Tmod1 after removal of the GST moiety as well as a few additional vector-derived amino acids at the carboxyl-terminal end of some of the Tmod1 fragments (Table I).Table IRecombinant Tmod1 fragments used in this studyTmod1 fragmentM raMolecular weights for purified recombinant proteins including linker and vector derived sequences.plAbs0.1%280Amino acid sequencebAmino acid sequences in boldface are the amino- and carboxyl-terminal sequences of Tmod1 or Tmod1 fragments. Amino acid sequences in italics at the amino terminal end are derived from the linker to GST. Tmod1 fragments 1-130, 1-156, 6-187, and 35-128 also had additional vector-derived sequences at their carboxyl terminal ends (in italics).FL 1-35941,5424.960.353GSPGISGGGGGILDS MSYRK... CRTGV1-32237,3474.760.392GSPGISGGGGGIRL MSYRK... HFTQQ1-23828,0994.760.476GSPGISGGGGGIRL MSYRK... TRSND1-29233,8834.720.395GSPGISGGGGGIRL MSYRK... DNQSQ6-18722,6684.460.477GSPGISGGGGGILDSM ELEKY... VEETL ELKLNSS1-15620,4034.620.530GSPGISGGGGGIL MSYRK... SSTIV GILDSMGRLELKLNS1-13016,2574.530.507GSPGISGGGGGIL MSYRK... AELCD ED35-12812,4594.620.457GSPGISGGGGILDSMD DELDP... SDAEL KLNSS35-35937,5215.070.322GSPGISGGGGGILDSM EELDP... CRTGV95-35930,6565.000.394GSPGISGGGGGILDSM AWIPK... CRTGV130-35926,5176.230.241GSPGISGGGGGIQ DIAAI... CRTGV160-34421,5246.830.178GSPGISGGGGGIG LNSVI... RKRRL160-35923,1087.920.166GSPGISGGGGGIG LNSVI... CRTGVa Molecular weights for purified recombinant proteins including linker and vector derived sequences.b Amino acid sequences in boldface are the amino- and carboxyl-terminal sequences of Tmod1 or Tmod1 fragments. Amino acid sequences in italics at the amino terminal end are derived from the linker to GST. Tmod1 fragments 1-130, 1-156, 6-187, and 35-128 also had additional vector-derived sequences at their carboxyl terminal ends (in italics). Open table in a new tab Transformed Escherichia coli (BL21(DE3)) cells were grown to an A 605 of 1.2–1.5 before inducing with 1 mm isopropyl-1-thio-β-d-galacto-pyranoside for 2 h at 37 °C, followed by lysis and purification using a modification of a previously described procedure (20Babcock G.G. Fowler V.M. J. Biol. Chem. 1994; 269: 27510-27518Abstract Full Text PDF PubMed Google Scholar). After affinity purification of the GST fusion protein on a glutathione column, the Tmod1 or fragment was released from the GST moiety by cleavage with thrombin for 30 min at 22 °C, using 12.5 units thrombin for each 2 liters of bacterial extract loaded onto 10–15 ml of beads. Thrombin concentration and cleavage times were adjusted for each fragment to maximize release from the GST moiety while minimizing internal proteolysis by thrombin. After elution from the beads, 50 units of recombinant hirudin (Calbiochem) were added to terminate thrombin cleavage and to prevent Tmod1 proteolysis by residual thrombin during subsequent purification steps. Tmod1 or fragments were dialyzed into 10 mm NaCl, 10 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol (DTT), 2.5 mm EDTA and purified by anion exchange chromatography on a Resource Q column (Amersham Biosciences), eluting with a 10–300 mm NaCl gradient in the above buffer, followed by chromatography on a Mono Q column (Amersham Biosciences), eluting with the same gradient. Tmod1 fragments 130–359, 160–359, and 160–344 with neutral or basic pI values were dialyzed into 20 mm MES, pH 6.5, buffer followed by purification on a Mono S (Amersham Biosciences) cation exchange column, eluting with a 10–300 mm NaCl gradient. Fractions containing Tmod1 or fragments were identified by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining, pooled, and dialyzed into 20 mm HEPES, pH 7.3, 80 mm KCl, 1 mm DTT, and 0.02% sodium azide and stored frozen at –80 °C. All of the fragments used in this study were soluble and well behaved, as expected from the previous biophysical studies, and yields ranged from 2 to 10 mg from 2 liters of bacterial extract, depending on the fragment. Before use in experiments, proteins were dialyzed and centrifuged for 30 min at 100,000 × g to remove minor amounts of aggregated material resulting from freezing and thawing, and their concentrations were redetermined. Protein concentrations were determined by light absorption at A 280 based on the extinction coefficients calculated from the amino acid composition of the purified fragments as described by Gill and Von Hippel (22Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar) using the ProtParam tool available on the World Wide Web at expasy.org/tools/protparam.html. The molecular weights, pI values, extinction coefficients, and amino- and carboxyl-terminal sequences for Tmod1 and Tmod1 fragments used in this study are summarized in Table I. Preparation of Actin, TM, and Gelsolin—Rabbit skeletal muscle actin was prepared from rabbit muscle acetone powder by the method of Spudich and Watt (23Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and further purified by gel filtration over Superose 6 column (Amersham Biosciences) as previously described (24Fowler V.M. Bennett V. J. Biol. Chem. 1984; 259: 5978-5989Abstract Full Text PDF PubMed Google Scholar). G-actin was stored by flash freezing in liquid nitrogen and defrosted as described (25Young C. Southwick F.S. Weber A. Biochemistry. 1990; 29: 2232-2240Crossref PubMed Scopus (37) Google Scholar). Before use, actin was dialyzed for 3–4 days at 4 °C into 4 mm Tris-HCl, pH 8.0, 0.1 mm CaCl2, 0.2 mm ATP, 1.0 m DTT, 0.02% sodium azide) followed by centrifugation for 30 min at 450,000 × g in a Beckman TLA 100.3 rotor to remove filaments and small oligomers that had formed during freezing and thawing. G-actin was stored on ice and used for up to 6 days. Pyrene-labeled actin was prepared as previously described (26Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (720) Google Scholar, 27Northrop J. Weber A. Mooseker M.S. Franzini-Armstrong C. Bishop M.F. Dubyak G.R. Tucker M. Walsh T.P. J. Biol. Chem. 1986; 261: 9274-9281Abstract Full Text PDF PubMed Google Scholar) and stored and treated as for unlabeled actin. Chicken skeletal muscle TM was prepared according to Smillie (28Smillie L.B. Methods Enzymol. 1982; 85: 234-241Crossref PubMed Scopus (227) Google Scholar) and stored as the lyophilized powder. Gelsolin was a gift from J. Bryan (Baylor College of Medicine, Dallas, TX) and was prepared as described (29Bryan J. J. Cell Biol. 1988; 106: 1553-1562Crossref PubMed Scopus (113) Google Scholar). Protein concentrations were determined for actin and gelsolin by light absorption, using E 290 = 24.9 mm–1 cm–1 and E 280 = 150 mm–1 cm–1, respectively, and for TM by Lowry's method, using bovine serum albumin as a standard. Actin Polymerization Measurements—Measurements of elongation rates at the pointed end were carried out using 8–12% pyrenyl-actin and gelsolin-capped actin filaments as nuclei for polymerization (11Weber A. Pennise C.R. Babcock G.G. Fowler V.M. J. Cell Biol. 1994; 127: 1627-1635Crossref PubMed Scopus (243) Google Scholar, 12Weber A. Pennise C.R. Fowler V.M. J. Biol. Chem. 1999; 274: 34637-34645Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In initial experiments, fluorescence was followed on a PerkinElmer 650S fluorometer and detected using a chart recorder (excitation = 366.5 nm, emission = 407 nm). Photobleaching was minimized by setting the excitation slit width to 2 nm and using a neutral density filter in the excitation light path (30Pollard T.D. Anal. Biochem. 1983; 134: 406-412Crossref PubMed Scopus (72) Google Scholar). In subsequent (most) experiments, fluorescence was followed using a Spex Fluoromax-3 fluorometer (excitation slit 0.5 nm, emission slit 5 nm) (Jobin Yvon Horiba, Edison, NJ), and data were collected using DataMax software. For assays in the absence of TM, gelsolin-actin seeds were prepared by copolymerizing 10 μm G-actin with 1 μm gelsolin in the presence of calcium (actin/gelsolin, 10:1) and used for up to 4 days. For each time course, Ca2+-actin was first converted to Mg2+-actin by incubation for 10 min at 20 °C in a buffer containing 10 mm imidazole, pH 7.0, 0.05 m MgCl2 and EGTA present at 2–3-fold in excess over the CaCl2 contributed by the actin buffer (25Young C. Southwick F.S. Weber A. Biochemistry. 1990; 29: 2232-2240Crossref PubMed Scopus (37) Google Scholar). Tmod1 or fragments and gelsolin/actin seeds were added in succession, and then polymerization was initiated by the addition of one-sixteenth volume of 16× polymerizing salts (1.65 m KCl, 33 mm MgCl2, 8.0 mm ATP, 3.3 mm CaCl2) and followed at 20 ° in the fluorometer. The final concentrations of components in the assays were as follows: 2.5 μm G-actin, 10–20 nm gelsolin/actin seeds, and Tmod1 or fragments as indicated in the figure legends. The buffer was 10 mm imidazole, pH 7.0, 0.1 m KCl, 2.0 mm MgCl2, 0.5 mm ATP, 0.2 m CaCl2, 1 mm DTT. Capping activities for Tmod1 and fragments were obtained from the initial elongation rates, measured directly from the slopes of the polymerization traces over the first 30 s to 1 min of polymerization. Rates in the presence of Tmod1 or fragments were divided by the rate for actin in the absence of Tmod1, giving a rate/control rate. The Kd values for full-length Tmod1 and each fragment were then calculated from the x intercept of a double reciprocal plot of 1/(1 – (rate/control rate)) versus 1/Tmod1 concentration. Although the absolute Kd values varied somewhat between different experiments, the relative differences in capping activities of the fragments as compared with full-length Tmod1 were very similar in each experiment. The variability for determination of the Kd values in these assays is most likely due to a variable efficiency of barbed end capping by gelsolin from one experiment to the next. Any free barbed ends will lead to a background of fluorescence increase due to barbed end elongation, which will lead to a falsely low readout for the pointed end-capping activity by Tmod1 and thus an apparently higher Kd value. Experiments measuring the effects of Tmod1 fragments on elongation rates in the presence of TM were generously carried out by Dr. Annemarie Weber at the University of Pennsylvania (Philadelphia, PA). Actin/gelsolin seeds (150:1) were copolymerized with TM, and additional TM was also added along with the Tmod1 or fragments in the polymerization mixtures to ensure that newly elongating filaments were also coated with TM (12Weber A. Pennise C.R. Fowler V.M. J. Biol. Chem. 1999; 274: 34637-34645Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Fluorescence was followed using a Photon Technology International fluorometer (Princeton, NJ). Elongation rates in the presence of TM and Tmod1 were measured from the flat portion of the curve after the inhibition of the polymerization rate by Tmod1 was maximal (e.g. see Fig. 4 in Ref. 12Weber A. Pennise C.R. Fowler V.M. J. Biol. Chem. 1999; 274: 34637-34645Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and Kd values were calculated as above. The effects of Tmod1 or fragments on spontaneous actin polymerization were examined by polymerizing 5 μm G-actin (8–10% pyrenyl-actin) in the presence or absence of Tmod1 or fragments. G-actin was first converted from Ca2+-actin to Mg2+-actin as described above, and then the indicated concentrations of Tmod1 or fragments were added (see Fig. 8 legend) followed immediately by one-tenth volume of 10× polymerizing buffer (1.0 m KCl, 20 mm MgCl2, 5.0 mm ATP, 50 mm EGTA) and incubation at 20 °C in the spectrofluorometer cuvette. The time course of fluorescence increase was monitored in a Fluoromax-3 fluorometer with the excitation slit at 0.25 nm and the emission slit at 10 nm to minimize photobleaching. The concentration of barbed ends was calculated from the elongation rate (measured by the rate of polymerization, where 50% of monomers were polymerized) using the equation, [Barbedends]=Rateofpolymerization(μM/s)/(k+[G-actin])(Eq. 1) where k + = 10 μm–1 s–1 (31Pollard T.D. J. Biol. Chem. 1986; 103: 2747-2754Google Scholar). Electrophoresis Procedures—Electrophoresis of proteins was on 12% SDS-polyacrylamide gels using a running gel pH of 8.6 (10Fowler V.M. J. Cell Biol. 1990; 111: 471-482Crossref PubMed Scopus (131) Google Scholar). Proteins were transferred to nitrocellulose, and blots were preheated at 65 °C in PBS followed by staining with Ponceau S to detect proteins (32Fowler V.M. J. Biol. Chem. 1987; 262: 12792-12800Abstract Full Text PDF PubMed Google Scholar) before labeling with a monoclonal antibody to tropomodulin (mAb9) (5Gregorio C.C. Weber A. Bondad M. Pennise C.R. Fowler V.M. Nature. 1995; 377: 83-86Crossref PubMed Scopus (160) Google Scholar), followed by rabbit anti-mouse IgG (Sigma) and detection by standard chemiluminescence procedures. CD Experiments—CD measurements of fragments 160–344 and 160–359 were performed on an Aviv model 62 D spectrophotometer in 100 mm NaCl, 10 mm sodium phosphate buffer, pH 6.5, at 10 °C. The secondary structure of these fragments were estimated using the programs CDNN (33Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 5: 191-195Crossref PubMed Scopus (1022) Google Scholar) and Selcon 1 (34Sreerama N. Woody R.W. Biochemistry. 1994; 33: 10022-10025Crossref PubMed Scopus (263) Google Scholar). Two different sets of standards were used with the Selcon program. The first contained 17 proteins where secondary structures were calculated as described by Kabsch and Sander (35Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar), and the second had 33 reference proteins where the secondary structures were calculated as described by Toumadje et al. (36Toumadje A. Alcorn S.W. Johnson Jr., W.C. Anal. Biochem. 1992; 200: 321-331Crossref PubMed Scopus (134) Google Scholar) as reviewed by Greenfield (37Greenfield N.J. Anal. Biochem. 1996; 235: 1-10Crossref PubMed Scopus (569) Google Scholar). Determination of the Free Energy of Folding of Peptide Fragments— The free energy of folding of fragments 160–344 and 160–359 were determined as described by Santoro and Bolen (38Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1609) Google Scholar), assuming that the free energy of folding is linearly dependent on the concentration of a chemical denaturant. In these experiments, a stock solution of 6 m guanidine HCl containing 10 μm peptide, 100 μm NaCl, and 10 mm sodium phosphate, pH 6.5, was used to titrate a 10 μm solution of each peptide, and the ellipticity (θobs) was recorded at each concentration of guanidine [Gn i ]. The data were fit to the equation, θobs={exp(-(ΔGo+M[Gni])/(RT))/(exp(-(ΔGo+M[Gni])/(RT))+1)}(θf-θu)+θu(Eq. 2) where ΔGo is the free energy of folding in the absence of denaturant, R is the gas constant, T is the temperature (Kelvin), θobs is the observed ellipticity at any concentration of guanidine HCl, [Gn i ], M is slope of the linear change in free energy as a function of [Gn i ], and θ f and θ u are the ellipticity of the fully folded and fully unfolded peptides at 10 °C. To solve the equation's initial values of ΔGo, M, θ f , and θ u were estimated, and the best fits to the data were evaluated using the Levenberg-Marquardt (39Marquardt D.W. J. Soc. Indust Appl. Math. 1963; 11: 431-441Crossref Google Scholar) algorithm implemented in SigmaPlot 8.0 (SpSS Inc., Chicago, IL). Recombinant Tmod1 Fragments—We chose to prepare a series of fragments from the chicken Tmod1 isoform (E-Tmod), since most of the previous structural and functional studies have been performed with this protein. Fig. 1 is a schematic depicting the locations of the fragments that we tested, with respect to the known structural features of Tmod1. In general, most fragments corresponded to the domain organization of Tmod1. Thus, the fragments 35–359, 95–359, and 130–359 all contained the compact, folded carboxyl-terminal domain but were missing increasingly greater amounts of the amino-terminal unstructured, TM-binding portion of Tmod1 (15Kostyukova A. Maeda K. Yamauchi E. Kri" @default.
• W2002802503 created "2016-06-24" @default.
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