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T Tao

Publications and source records attributed to T Tao.

At least 109 records · Page 6Linked to original sources
PubMed1990

Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

Actins↗
PubMed1989

Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements.

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.

Animals↗
PubMed1989

Sequence, internal homology and high-level expression of the gene for a DNA-(cytosine N4)-methyltransferase, M.Pvu II.

The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.Pvu II. Specifically, the composite Ptac promoter was inserted 5' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB laclq strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.Pvu II accumulated to the point of comprising over 10% of the total soluble protein.

Amino Acid Sequence↗
PubMed1988

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T.

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Cloning, sequencing and expression of a full-length rabbit fast skeletal troponin-C cDNA.

A full-length cDNA coding for troponin-C isolated from an adult rabbit fast skeletal muscle library has been sequenced and expressed in an E. coli host. The amino acid sequence derived from the coding region agrees with the published protein sequence except that the first two residues are reversed. The expressed protein was found to be identical to purified rabbit skeletal troponin-C based on electrophoretic mobilities in polyacrylamide gels containing either SDS or urea, and on immunoblotting. These results establish that our troponin-C cDNA clone is suitable for site-directed mutagenesis studies on the structure and function of troponin-C.

Animals↗
PubMed1988

Transmission of the Ca2+-regulatory signal in skeletal muscle thin filaments.

Studies dealing with some aspects of the thin-filament based regulatory mechanism of striated muscle are discussed. A key event is a Ca2+-induced shift of TnI from a binding site on actin to TnC, as indicated by optical and crosslinking experiments. The regions concerned involve residues 1-12 in actin, residues 96-116 in TnI, and residues 89-100 in TnC. These changes in interaction are accompanied by changes in TnC and TnI that occur on a millisecond time scale which is consistent with their being part of the regulatory processes underlying regulation in vivo.

Actin Cytoskeleton↗
PubMed1987

Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C.

We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. Upon photolysis, covalent cross-links were formed between TnC and TnI [Tao, T., Scheiner, C.J., & Lamkin, M. (1986) Biochemistry 25, 7633-7639]. The cross-linked heterodimer was digested with cyanogen bromide, pepsin, and chymotrypsin into progressively smaller cross-linked peptides, which were purified by HPLC and then characterized by amino acid analysis and sequencing. We obtained a fraction from the initial CNBr digest that contained the expected peptide CB9 (residues 84-135) of TnC, cross-linked mainly to CN4 (residues 96-116), the "inhibitory region" of TnI. The peptides CN1 and CN3 of TnI were also detected in this fraction, but their molar ratios (compared to CB9) were only about 0.15 each, compared to 0.60 for CN4. Sequence analyses of fractions obtained after peptic and chymotryptic digests of the cross-linked CNBr fraction confirmed that CB9 and CN4 were the major cross-linked species. Quantitative analysis of sequencer results indicated that the residues in TnI that appeared to be most highly cross-linked to Cys-98 of TnC were Arg-108 and Pro-110, and to a lesser extent Arg-103 and Lys-107. These findings are consistent with previous studies on interactions between TnI and TnC and provide, for the first time, direct information on the identities of proximate amino acids in the two proteins.

Amino Acid Sequence↗
PubMed1987

pH-dependent structural transition in rabbit skeletal troponin C.

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.

Animals↗
PubMed1986

Site-specific photo-cross-linking studies on interactions between troponin and tropomyosin and between subunits of troponin.

We have used the sulfhydryl-specific heterobifunctional photo-cross-linker 4-maleimidobenzophenone (BP-Mal) to study the interactions of rabbit skeletal tropomyosin with troponin and of the troponin subunits with each other. We found that alpha,alpha-tropomyosin specifically labeled at Cys-190 with BP-Mal photo-cross-links with all three subunits of troponin with decreasing cross-linking yields in the order of troponin T, troponin I, and troponin C. There was no apparent Ca2+ dependence in the cross-linking yields. In separate experiments, we found that troponin C labeled specifically at Cys-98 with BP-Mal photo-cross-links to both troponin I and troponin T in the two binary complexes, as well as in the ternary complex. Again, no Ca2+-dependent changes in the cross-linking yields were detectable. These results are in general agreement with the picture that troponin I and troponin T are in close contact with troponin C near its Cys-98 and that all three troponin subunits are in the proximity of Cys-190 of tropomyosin.

Actins↗
PubMed1986

Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.

Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified either with 1,5-IAEDANS (N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine), as energy transfer donor, or with IAF (5-(iodoacetamido)fluorescein) or DABMI (4-dimethylaminophenylazophenyl-4'-maleimide), as energy transfer acceptor. The sulfhydryl groups on these light chains are located at different positions within the regulatory light-chain primary sequence; this enables one to probe a variety of locations, with respect to regulatory light-chain topology, on each myosin head. These independently modified regulatory light chains were added back to desensitized scallop myosin under a variety of conditions, including biphasic re-addition, the aim being to maximize the number of interhead energy transfer couples present. The efficiency of energy transfer was determined on the same samples by both steady-state and time-decay techniques. Results obtained by these two techniques were in good agreement with each other and indicated that the efficiency of energy transfer did not exceed 20% in any of the hybrids studied. Transfer efficiencies were invariant, irrespective of the presence or absence of MgATP, calcium or actin, either separately or in combination. Results using heavy meromyosin at low ionic strength were identical. It is shown that these results, in conjunction with the results of recent crosslinking studies performed on comparable myosin hybrids, may place certain restrictions on the configurations of the two heads of myosin.

Animals↗
PubMed1985

The conformation of the C-terminal region of actin: a site-specific photocrosslinking study using benzophenone-4-maleimide.

The site-specific photocrosslinker, benzophenone-4-maleimide, was used to label G-actin specifically at Cys-374, the penultimate residue from the C terminus. The resultant BP-G-actin was polymerized to form BP-F-actin, and both forms of actin were irradiated to activate the benzophenone moiety. We found that for BP-F-actin both intersubunit and intrasubunit photocrosslinks were formed. For BP-G-actin only a small amount of an internally photocrosslinked species was formed. These findings suggest that in the F-actin polymer, the C-terminal peptide is localized in a region between neighboring subunits. In contrast, in the G-actin monomer, the C-terminal peptide is relatively distant from the surface of the molecule.

Actins↗
PubMed1984

Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits.

Fluorescence lifetime and acrylamide quenching studies were carried out to characterize the interactions between the subunits of troponin under various conditions of metal ion binding. Troponin C was labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. In the presence of Ca2+, the fluorescence decay of labeled troponin C (TnC*) was monoexponential, lifetime tau = 15.5 ns and quenching rate constant kq = 2.97 X 10(8) M-1 s-1. In the absence of Ca2+, the decay was resolvable into a major component with tau = 11.9 ns and a minor component with tau = 20.5 ns, with corresponding values of kq = 4.80 X 10(8) and 0.66 X 10(8) M-1 s-1, respectively. Upon the binding of either troponin I (TnI) or troponin T (TnT) in the presence of Ca2+, tau increased to approximately 18 ns, and kq decreased to approximately 0.8 X 10(8) M-1 s-1. For the Ca2+ form of the TnC*-TnI-TnT ternary complex, values of tau = 17.6 ns and kq = 1.73 X 10(8) M-1 s-1 were obtained. These values did not vary significantly when Ca2+ was removed, or when Mg2+ replaced Ca2+. These findings were interpreted as follows: the region around Cys-98 of TnC* adopts a looser conformation upon the removal of Ca2+ from the high-affinity sites. Both TnI and TnT bind to TnC* in the region containing Cys-98. The probe is shielded from the solvent to a greater extent in the binary complexes than in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Crosslinking of tropomyosin to myosin subfragment-1 in reconstituted rabbit skeletal thin filaments.

Rabbit skeletal tropomyosin was labeled with the bifunctional photoactivatable crosslinker N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. After irradiating the rigor complex composed of myosin subfragment-1, crosslinker-labeled tropomyosin, and F-actin, a crosslinked product was formed. This product was identified as a 1:1 adduct of tropomyosin and subfragment-1. This finding is in support of recent structural studies which suggest that tropomyosin and subfragment-1 are in close proximity to each other, and may be relevant to the mechanism of thin filament regulation.

Actins↗
PubMed1983

Tropomyosin-troponin and tropomyosin-actin interactions: a fluorescence quenching study.

Rabbit skeletal alpha alpha-tropomyosin was specifically labeled at Cys-190 with the fluorescent probe N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)ethylenediamine (1,5-IAE-DANS). The fluorescence decay of the resultant AE-DANS-labeled alpha alpha-tropomyosin (Tm) was monoexponential with a lifetime of 13.55 ns. When acrylamide was used as the quencher, the apparent Stern-Volmer quenching constant Ksv' for Tm was measured to be 5.78 M-1 and the quenching rate constant kq to be 3.20 X 10(8) M-1 s-1. The presence of troponin reduced the magnitude of Ksv' to 4.14 M-1 and induced the appearance of a second decay component. This second component had an amplitude of approximately 20% of the total intensity, a lifetime of approximately 20 ns, and a kq of 4.5 X 10(-7) M-1 s-1. Similarly, the presence of F-actin induced the appearance of a minor longer lived decay component with a decreased kq. On the basis of the increase in the lifetime and the decrease in kq, the appearance of the long-lived decay component was interpreted to be due to troponin or actin interacting with Tm near the Cys-190 site in both cases. Our results further suggest that the label was capable of equilibrating between an exposed hydrophilic environment on the surface of Tm and a buried hydrophobic environment at the troponin-Tm or actin-Tm interaction interfaces.

Acrylamide↗
PubMed1982

The distance between the high affinity sites of troponin-C measured by interlanthanide ion energy transfer.

Trivalent lanthanide ions are known to be good substituents for Ca2+ at all four calcium-binding sites of rabbit skeletal troponin-C (TnC). In particular, the visible luminescence of terbium ions bound at the two high affinity sites can be excited via a tyrosine residue. We have carried out energy transfer measurements using Tb3+ as the donor and a number of lanthanide ions as acceptors in order to measure the distance between the two high affinity sites in TnC. Luminescence decay measurements showed that, in the absence of acceptors, TnC-bound Tb3+ decays with a single lifetime of 1.31 ms. In the presence of a good acceptor such as Nd3+, the decay was resolvable into two components of roughly equal amplitude. The first was the same in lifetime as that of TnC-bound Tb3+ alone; the second has a shorter lifetime, presumably due to interlanthanide ion energy transfer. From these lifetimes and published critical transfer distances (Horrocks, W. DeW., Jr., Rhee, M.-J., Snyder, A.P., and Sudnick, D.R. (1980) J. Am. Chem. Soc. 102, 3650-3652), we obtained a distance of 0.92 nm between the two high affinity sites. This distance is consistent with the fact that the two high affinity sites have been located in the COOH-terminal half of TnC and with the proposal that each half of TnC is homologous in structure to parvalbumin (Kretsinger, R. H., and Barry, C. D. (1975) Biochim. Biophys. Acta 405, 40-52).

Animals↗