PubMed Health⌕ Search

Biomedical subjects

H C Cheung

Publications and source records attributed to H C Cheung.

At least 37 records · Page 2Linked to original sources
PubMed1997

Phosphorylation-induced distance change in a cardiac muscle troponin I mutant.

Phosphorylation of two adjacent serine residues in the unique N-terminal extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments. To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine: (1) a full-length cTnI mutant (S5C/C81I/C98S) and (2) a truncated cTnI mutant (S9C/C50I/C67S) in which the N-terminal 32 amino acid residues were deleted. We determined the apparent binding constants for the complex formation between IAANS-labeled cardiac troponin C (cTnC) and the two cTnI mutants. The affinities of the cTnC for the truncated cTnI mutant were: (1) 1.5 x 10(6) M(-1) in EGTA, (2) 28.9 x 10(6) M(-1) in Mg2+, and (3) 87.5 x 10(6) M(-1) in Mg2+ + Ca2+. These binding constants were approximately 1.4-fold smaller than the corresponding values obtained with the full-length cTnI mutant, suggesting a very small contribution of the N-terminal extension to the binding of cTnI to cTnC. Cys-5 in the full-length cTnI mutant was labeled with IAANS, and the distribution of the separation between this site and Trp-192 was determined by analysis of the efficiency of fluorescence resonance energy transfer from Trp-192 to IAANS. The following mean distances were obtained with the unphosphorylated full-length mutant: 44.4 A (cTnI alone), 48.3 A (cTnI + cTnC), 46.3 A (cTnI + cTnC in Mg2+), and 51.6 A (cTnI + cTnC in Mg2+ + Ca2+). The corresponding values of the mean distance determined with the phosphorylated full-length cTnI mutant were 35.8, 36.6, 34.8, and 37.3 A. The phosphorylation of cTnI reduced the half-width of the distribution from 9.5 to 3.7 A. Similar but less pronounced decreases of the half-widths were also observed with the phosphorylated cTnI complexed with cTnC in different ionic conditions. Thus, phosphorylation of cTnI resulted in a decrease of 9-12 A in the mean distance between the sites located at the N- and C-terminal portion of cTnI. Our results indicate that phosphorylation elicits a change in the conformation of cTnI which underlies the basis of the phosphorylation-induced modulation of cTnI activity.

Animals↗
PubMed1997

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.

Acrylamide↗
PubMed1997

Disparate fluorescence properties of 2-[4'-(iodoacetamido)anilino]-naphthalene-6-sulfonic acid attached to Cys-84 and Cys-35 of troponin C in cardiac muscle troponin.

Two monocysteine mutants of cardiac muscle troponin C, cTnC(C35S) and cTnC(C84S), were genetically generated and labeled with the fluorescent probe 2-[4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-84 and Cys-35, respectively. Cys-84 is located on helix D in the regulatory N-domain, and Cys-35 is at the -y position of the inactive 12-residue loop of site I. These labeled mutants were studied by a variety of steady-state and time-resolved fluorescence methods. In the absence of divalent cation, the fluorescence of the attached IAANS indicated an exposed environment at Cys-35 and a relatively less-exposed environment at Cys-84. The binding of Ca2+ to the single regulatory site elicited a large enhancement of the emission of IAANS attached to Cys-84, but only marginal fluorescence changes of the probe at Cys-35. Upon reconstitution of the labeled cTnC mutants with troponin I and troponin T to form the three-subunit troponin, the fluorescence of IAANS-Cys-84 in apo-troponin was spectrally similar to that observed with the Ca(2+)-loaded uncomplexed cTnC mutant. Only very moderate changes in the fluorescence of IAANS-Cys-84 were observed when the regulatory site in reconstituted troponin was saturated. The exposed Cys-35 environment of the uncomplexed cTnC mutant became considerably less exposed and less polar when the mutant was incorporated into apo-troponin. In contrast to the Cys-84 site, saturation of the regulatory site II by Ca2+ in reconstituted troponin resulted in a reversal of the environment of the Cys-35 site toward a more exposed and more polar environment. These results indicated involvement of the inactive loop I in the Ca2+ trigger mechanism in cardiac muscle. The fluorescence of IAANS at both Cys-84 and Cys-35 was sensitive to phosphorylation of cTnl in reconstituted troponin, and the sensitivity was observed with both apo-troponin and Ca(2+)-loaded troponin.

Animals↗
PubMed1996

Structural studies of kinesin-nucleotide intermediates.

We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.

Adenosine Diphosphate↗
PubMed1996

Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84.

Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain an insight about the nature of Ca(2+)-induced conformational changes in the N-domain regulatory region of cTnC. Changes in the experimental emission spectra, quantum yields, and excited-state lifetimes suggested that bound Ca2+ at the single regulatory site induced a less polar microenvironment for both probes attached to Cys-84. However, a twofold increase in the bimolecular collisional quenching constant was observed for both probes in the presence of activator Ca2+, indicating an increased exposure of the probes to solvent. These data were interpreted with reference to the origins of the observed Stokes' shifts. In the apo and 2Mg states of cTnC, the attached probes were partially shielded by helices B and C, and their excited-states were highly quenched in the tertiary structure through strong interactions of a dipolar nature with neighboring amino-acid side chains. In the 3Ca state, these interactions were disrupted so that nonradiative decay processes were suppressed and radiative processes were enhanced, leading to the observed increases in quantum yields and lifetimes and blue-shifts of the emission spectra. As the disruption of internal quenching resulted from separation of helices B and C from helix D, the attached probes became more accessible to solvent and experienced increases in the rate of collisions with external molecules in the solvent. Although this increased exposure to solvent would lead to suppression of radiative decay processes, this effect apparently was overcompensated by the effect of elimination of internal quenching. The present results are consistent with a Ca(2+)-induced open conformation of the N-domain in cTnC.

2-Naphthylamine↗
PubMed1996

Equilibrium studies of kinesin-nucleotide intermediates.

We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule.

Adenosine Diphosphate↗
PubMed1996

Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle.

We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C by measuring the rates of calcium-mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys-84 with the fluorescent probe 2(-)[4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At 4 degrees C, the binding kinetics determined in the presence of Mg2+ was resolved into two phases with positive amplitude, which were completed in less than 100 ms. The rate of the fast phase increased linearly with [Ca2+] reaching a maximum of approximately 590 s-1, and that of the slow phase was approximately 100 s-1 and did not depend on Ca2+ concentration. Dissociation of bound Ca2+ from the regulatory site occurred with a rate of 102 s-1, whereas the dissociation from the two high affinity sites was about two orders of magnitude slower. These results are consistent with the following scheme for the binding of Ca2+ to the regulatory site: [formula: see text] where the asterisks denote states with enhanced fluorescence. The apparent second-order rate constant for calcium binding is Kok1 = 1.4 x 10(8) M 1 s-1. The two first-order transitions occur with observed rates of k1 + kappa-1 approximately 590 s-1 and kappa 2 + kappa-2 approximately 100 s-1, and the binding of Ca2+ to the regulatory site is not a simple diffusion-controlled reaction. These transitions provide the first information on the rates of Ca(2+)-induced conformational changes involving helix movements in the regulatory domain.

Animals↗
PubMed1996

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.

Animals↗
PubMed1995

Peptide mimetics of thyrotropin-releasing hormone based on a cyclohexane framework: design, synthesis, and cognition-enhancing properties.

The design and synthesis of peptide mimetics of thyrotropin-releasing hormone (TRH) in which the peptide backbone is entirely replaced by a cyclohexane framework are described. The cis-1,3,5-trisubstituted ring was expected to permit key pharmacophoric groups to adopt conformations consistent with proposed bioactive conformations of the peptide. Compounds were synthesized by a stereoselective synthesis starting from L-glutamic acid. In a behavioral model of cognition in which TRH is active, the mimetics are potent, active compounds, exhibiting oral activity. One analog (26, (1S,3R,5(2S),5S)-5-[[5-oxo-1-(phenylmethyl)-2-pyrrolidinyl]-methyl]-5- [(1H-imidazol-5-yl)methyl]cyclohexaneacetamide) was radiolabeled for binding studies and evaluated in other binding assays and pharmacological tests. Competition binding of 26 vs [3H]MeTRH to rat brain slices suggests a two-site model for ligand binding with IC50's of 1 microM and 3 mM. Direct binding of [3H]-26 shows a biphasic curve with IC50's of 80 and 49 microM, respectively. Further studies would be needed to establish a link between the novel binding site(s) and the behavioral activity of 26 and TRH analogs.

Amino Acid Sequence↗
PubMed1995

Internal movement in myosin subfragment 1 detected by fluorescence resonance energy transfer.

We have determined intersite distances from Cys374 of actin to Cys707 (SH1) and Cys697 (SH2) of myosin subfragment 1 (S1) in actosubfragment 1 (A.S1) by fluorescence resonance energy transfer for rigor complex A.S1 and complexes containing bound ADP and ADP plus orthovanadate (Vi), A.S1.ADP, and A.S1.ADP.Vi. A single energy acceptor (4-dimethylaminophenylazophenyl-4'-maleimide, DABMI) was attached to Cys374, and two different energy donors [(5-(iodoacetamideothyl)aminonaphthalene-1-sulfonic acid (IAEDANS) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)] were each attached to SH1 and SH2 for the distance determination. The two sites SH1 and SH2 of S1 were approximately equidistant (ca. 45 A) from actin Cys374 in rigor A.S1 when MIANS was the energy donor attached to the two thiols. The Cys374-SH1 distance decreased by 7-8 A in the presence of ADP plus Vi, but the distance Cys374-SH2 was essentially unaltered under identical conditions. Slightly different but similar distance results were obtained with AEDANS as energy donor. If the structure of actin monomer in A.S1 is assumed to be rigid [Miki, M. (1991) Biochemistry 30, 10878-10884], the present results indicate that MgADP plus Vi induced a movement of SH1 toward the actin site and that SH2 was insensitive to saturation of the active site pocket of S1 and relatively immobile. These results suggest that during the steady-state hydrolysis of ATP or in the weak-binding state of actomyosin, the short helical segment of S1 heavy chain containing SH1 moves closer to the COOH-terminal end of actin, while the adjacent helical segment containing SH2 remains stationary. The emission spectrum of MIANS attached to SH2 experienced a large red spectral shift (6-10 nm) in the presence of MgADP, MgADP + Vi, MgADP + beryllium fluoride, and ATP. A crude model of S1 based on the C alpha coordinates suggests that SH2 is located in a hydrophobic cage surrounded by three hydrophobic residues. Reorientation of one of these side chains could expose SH2 to the solvent. The observed red spectral shift of MIANS attached to SH2 could be explained by such a nucleotide-induced exposure, and this explanation would be consistent with the interpretation that SH2 is stationary.

Actins↗
PubMed1994

Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin.

The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain. The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) (AMPPNP). Fluorescence lifetime studies using 1-N6-ethenoadenosine plus beryllium fluoride reveal two components for both (10 S)- and (6 S)-myosins, with little difference in the values of these lifetimes, their fractional amplitudes, or solute accessibilities. Anisotropy decay studies of myosin-mant nucleotide complexes demonstrate that the rotational correlation time for (10 S)-myosin is nearly 4-fold longer than that for (6 S)-myosin. Qualitatively similar results were obtained with a 5-[[[2(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescent probe attached to the regulatory light chain. Mant AMPPNP can be trapped in the active site by (10S)-myosin. Actin accelerates this release rate by 40-50-fold. These studies reveal: 1) reduction in nucleotide release rate by converting (6S) to (10S)-myosin is not due to a reduction in solute accessibility of the nucleotide 2) the heads in (10 S)-myosin are rigidly attached to the rest of the molecule, while in (6 S)-myosin, they have segmental flexibility, 3) regulatory light chain phosphorylation mimics the effect of high salt in enhancing segmental flexibility of the myosin heads, and 4) actin can induce the unfolding of (10 S)-myosin in the absence of regulatory light chain phosphorylation.

Acrylamide↗
PubMed1994

Tropomyosin length and two-stranded F-actin flexibility in the thin filament.

In muscle thin filaments, each tropomyosin molecule is considered to be a rope-like structure that winds along the filament in contact with seven consecutive actin monomers on the same strand of the two-stranded actin helix. Taking into account the head-to-tail overlap of the tropomyosin molecule, the effective length of this "rope" is about 405 angstrum, which is believed to be conserved. Tropomyosin appears to be neither extensible nor compressible in its axial direction, although it may possess much flexibility in the transverse direction. During the "maximally on" state, characterized by the presence of Ca2+ and the strong binding between actin and myosin subfragment 1, the following conditions are thought to occur: the motion and associated flexibility of tropomyosin are reduced; the actin filament flexibility increases; a maximum number of equivalent tropomyosin binding sites on actin are concurrently saturated; and the tropomyosin molecule maintains an average thin filament radius of 38 to 40 angstrum. Under these potentiated conditions, the length of tropomyosin can be used to determine the limits on the underlying "cumulative angular disorder" of the actin filament with which it interacts. Our calculations show that only a small amount (approximately 1 to 3 degrees) of this type of actin monomer rotational disorder is possible at this stage of the contractile cycle, unless the length of the tropomyosin molecule is increased substantially between the head-to-tail joints. However, if the dominant type of F-actin rotational flexibility is between two relatively rigid actin strands (the lateral slipping/rotational offset model), all of the above actin-tropomyosin interactions can be completely and easily accommodated. We also discuss the implications of an interdomain hinge in G-actin and the possibility that there may be fewer than seven equivalent sites on actin that are saturated by tropomyosin concurrently.

Actin Cytoskeleton↗
PubMed1994

Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle.

The interaction of troponin I (CTnI) with troponin C (CTnC) from bovine cardiac muscle was studied using CTnC modified at Cys35 and Cys84 with the fluorescent probe 2-[(4'-iodoacetamido)-anilino]naphthalene-6-sulfonic acid (CTnCIAANS). The association constant for complex formation between the two proteins was determined at 20 degrees C in 0.4 M KCl, 1 mM DTT, 1 mM EGTA, and 25 mM MOPS, pH 7.2. In the presence of EGTA, Mg2+, and Ca2+ these constants were 1.46 x 10(7), 4.1 x 10(7), and 12.7 x 10(7) M-1, respectively, with corresponding free energy values of -9.62, -10.23, and -10.88 kcal mol-1. The CTnI-CTnCIAANS complex was stabilized by -0.61 kcal when the two Ca/Mg sites of CTnCIAANS were saturated with Mg2+ and by -1.26 kcal when all three Ca2+ sites were occupied by Ca2+. These results suggest that calcium activation in cardiac muscle may be accompanied by a coupling free energy of -0.65 kcal. This value is a factor of 4 smaller than the value previously determined, using a similar method, for the (troponin I).(troponin C) complex from skeletal muscle [Wang, C.-K., & Cheung, H.C. (1985) Biophys. J.48, 727-739]. Since CTnC has only one Ca(2+)-specific site and troponin C from skeletal muscle has two such sites, the present result is a factor of 2 smaller than that for the skeletal complex on the basis of a single specific site. Phosphorylation of CTnI by 3',5'-cyclic AMP-dependent protein kinase resulted in a decrease of the association constants by a factor of 2.5-3.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Vanadate-induced changes in myosin subfragment-1 from cardiac muscle.

The interaction between myosin subfragment-1 from bovine cardiac muscle (CS1) and 1,N6-ethenoadenosine diphosphate (epsilon ADP) was studied using steady-state and time-resolved fluorescence methods. The binding constant was found to be 1.52 x 10(6) M-1 at pH 7.5 and 5 degrees C. The intensity decay of epsilon ADP bound to CS1 was resolved into two components over a narrow range of temperatures. The long component was about 22 ns and the short component was between 5 and 7 ns, with fractional amplitudes of about 0.6-0.7 for the long component and 0.3-0.4 for the short component. These data suggest a two-state temperature-sensitive transition of the CS1.epsilon ADP complex. In the presence of orthovanadate (Vi) at 5 degrees C, the decay time of the long component was little affected, whereas the short decay time increased by over 3 ns and the fractional amplitude of the long component decreasing by a factor of 2 to about 0.3 and that of the short component increasing to 0.7. The anisotropy decay of bound epsilon ADP was monoexponential regardless of whether vanadate was present. The recovered single rotational correlation time was 110 ns in the absence of vanadate and 79 ns in the presence of vanadate. The decrease in correlation time suggests an increase in molecular symmetry of the CS1.epsilon ADP.Vi complex. The results are in agreement with previous results obtained from skeletal S1 and indicate that S1 from both isoforms of myosin experiences similar vanadate-induced changes in its hydrodynamic shape. Since the ternary vanadate complex is a stable analogue of the S1.ADP.Pi state, the ligand-induced change in hydrodynamic shape of S1 may be related to the conformational change which myosin head experiences during the ATPase cycle and this change in myosin could be a structural basis for force generation in striated muscle.

Adenosine Diphosphate↗
PubMed1993

Rotational dynamics of skeletal muscle troponin C.

Upon excitation by 280 nm, the intensity decay of the 2 tyrosine residues (residues 10 and 109) of rabbit skeletal muscle troponin C is resolved into three components. The anisotropy decay in the absence of divalent cation is biphasic with a short correlation time of 0.67 ns and a long correlation time of 9.23 ns. The limiting anisotropy is 0.225, considerably lower than the value expected for immobilized tyrosine. Upon excitation by 290 nm, the anisotropy decay is also biphasic, and the limiting anisotropy increases to 0.274. The recovery of anisotropy by excitation at a wave-length near the red edge of the tyrosine absorption spectrum is evidence of fluorescence resonance energy transfer between the two tyrosines. For energy transfer to occur, the average separation between the 2 tyrosines is unlikely much larger than the Förster distance Ro, congruent to 10 A, and this close proximity of the residues would require a highly distorted dumbbell shape of troponin C in solution. These results are consistent with a flexible central helix, which either has a segmental flexibility with large amplitude or results in a spectrum of conformations including those in which the two globular domains are in a very close proximity.

Animals↗
PubMed1993

Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.

Actins↗
PubMed1992

The kinetics of a two-state transition of myosin subfragment 1. A temperature-jump relaxation study.

Temperature-jump measurements were carried out on myosin subfragment 1 (S1) labeled at Cys-707 with 5-(iodoacetamido)fluorescein (S1-AF). The relaxation was monitored by following the increase in the fluorescence intensity of the attached probe after a jump of 5.8 degrees C. A single relaxation process was observed over a range of final temperatures, and the relaxation time decreased from 16.69 ms at 15 degrees C to 3.91 ms at 27 degrees C. The relaxation results are interpreted in terms of a two-state transition: (S1-AF)L K+ in equilibrium with K- (S1-AF)H, and the observed single relaxation time (tau) equals l/(k(+) + k-). The individual first-order rate constants, k+ and k-, were calculated from tau and the equilibrium constant previously determined. The activation energy was 21.9 kcal/mol for the forward reaction and 9.3 kcal/mol for the reverse reaction, corresponding to an enthalpy value of 12.6 kcal/mol for the two-state transition. The results provide, for the first time, direct kinetic evidence of a two-state transition of S1 in the absence of bound nucleotide, and support a two-state model of unliganded myosin subfragment 1.

Hot Temperature↗