Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 NMR

Shriver, John W.; Sykes, Brian D.

doi:10.1021/bi00541a033

Cited by 51 publications

(23 citation statements)

References 33 publications

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“…These data have been interpreted as indicating that the myosin heads exist as an equilibrium mixture of two conformational states even when they do not interact with nucleotide or actin [17]. Such a possibility has also been considered on theoretical grounds [18] and has found experimental support from the effects of nucleotides and temperature on the I9F-NMR spectrum of a fluorine probe attached to the SH1 thiol group in rabbit skeletal muscle S1 [19, 201. It has been suggested that the conformational distortions underlying the myosin head alternation between the weak and strong actin binding states in the crossbridge cycle [21] resolve themselves into nucleotide-and actininduced shifts in the equilibrium between the two intrinsic states of the head [17-201.…”

mentioning

confidence: 99%

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

Rędowicz

Sobieszek

Strzelecka‐Gołaszewska

1990

European Journal of Biochemistry

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It was previously shown that tryptic digestion of subfragment 1 (Sl) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH,-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25"C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH,-terminus is involved in the communication between the sites of nucleotide and actin binding.We also show here for the first time that the S1-S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain-produced S1 devoid of the regulatory light chain) is also temperaturedependent but insensitive to nucleotides and actin. It is suggested that the temperature-dependent alteration in the flexibility of the head-rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J . Mol. Biol. 182,[271][272][273][274][275][276][277][278][279] Biochem. 177,, may contribute to the temperature dependence of some steps in the cross-bridge cycle.Current models of the cross-bridge action in contracting muscle place the force-generating structural transition alternatively in the myosin head-rod junction [I, 21, in Conformational changes in the myosin molecule that might be relevant to the cross-bridge action have been extensively studied using a variety of experimental approaches (for review see [8 -1 I]). One of them involves examination of the effects of nucleotides and actin binding on the pattern and kinetics of proteolytic degradation of myosin and its subfragments. Limited digestion with trypsin of the chymotryptic subfragment 1 (Sl) of rabbit skeletal muscle myosin produces three heavy chain fragments, of 27 kDa, 50 kDa, and 20 kDa, representing the NH2-terminal, central, and COOH-terminal heavy chain domains, respectively. The binding of nucleotides to S1 results in the exposure of a new tryptic cleavage site in the NH2-terminal fragment, about 5 kDa from its NH2-terminus, and promotes an additional cleavage of the central fragment near its COOH-terminus [13 -171. These nucleotide- promoted cleavages are suppressed after formation of a ternary F-actin-S1 -nucleotide complex [15, 161. We have previously shown that the same site near the NH,-terminus is rendered susceptible to trypsin at low temperature in the absence of nucleotide, and that this low-temperature-promoted cleavage is also suppressed by actin. These data have been interpreted as indicating that the myosin heads exist as an equilibrium mixture of two conformational states even when they do not interact with nucleotide or actin [17]. Such a possibility has also been cons...

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mentioning

confidence: 99%

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

Rędowicz

Sobieszek

Strzelecka‐Gołaszewska

1990

European Journal of Biochemistry

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“…However, the results obtained with various techniques differ with regard to the direction of the temperatureand nucleotide-induced alterations. The changes in the hydrogen-exchange rate of tryptophan residues [57], ultraviolet absorption [56], and EPR spectra of spin-labeled heavy meromyosin or myosin [35, 581 that occur on elevation of temperature were qualitatively similar to those induced by MgADP or MgAdoPP[NH]P. The 19F-NMR spectra of a fluorine probe attached at the SH1 thiol group indicated, on the contrary, that the low-temperature state of S1 is identical to that predominating in the presence of MgADP or MgAdoPP[NH]P at 25°C [55]. Our results are in accord with the latter observation.…”

Section: Discussionmentioning

confidence: 99%

“…The results have been interpreted as indicating that the myosin head itself exists in two conformational states in equilibrium which depends on ambient factors and is perturbed by the binding of nucleotides [36, 37, 52, 541. Evidence supporting this conclusion has been provided by studies of 19F-NMR spectra of a fluorine-containing probe attached to the SH1 thiol group of S1 [55] and ultraviolet absorption [56], hydrogen-deuterium exchange [57] and spin-label studies on heavy meromyosin [58]. Geeves et al [59] and Shriver [54] have recently reviewed and discussed kinetic evidence for two types of ternary complexes of myosin with nucleotide and actin which differ in the affinity between myosin and actin.…”

mentioning

confidence: 99%

Conformational transitions in the myosin head induced by temperature, nucleotide and actin

Rędowicz

Szilágyi

Strzelecka‐Gołaszewska

1987

European Journal of Biochemistry

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Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (Sl) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides.The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its P,yimido analog AdoPP[NH]P) at 25 "C were observed when the digestion was carried out at 0°C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoPP[NH]P at 0°C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.It is generally accepted that contraction of muscle results from a relative sliding movement of the myosin thick filaments past the actin thin filaments driven by a cyclic interaction of myosin heads or cross-bridges with actin, each cycle being coupled to hydrolysis of one molecule of ATP [l]. However, the detailed mechanism of conversion of the free energy of ATP hydrolysis into mechanical energy remains a subject of debate. A prevailing model suggested by H. E. Huxley [l] and by A. F. Huxley and Simmons [2] assumes that force is generated by an active change in the angle of attachment of myosin heads to actin filaments by rotation about a flexible joint between the head (myosin subfragment-1) and the neck region (subfragment-2) of the cross-bridge. An alternative proposal is the rotation of one portion or domain of the head relative to the other portion which remains rigidly attached to actin [3,4]. The latter possibility is in better agreement with the evidence from recent X-ray diffraction studies on muscle [5 -71 and with fluorescence polarization [8] and EPR studies [9] on glycerinated muscle fibers.The possible existence of domains within the myosin head has been inferred from limited proteolysis studies on myosin subfragment-1. The heavy chain of chymotryptic S1 of myosin from rabbit skeletal muscle is split by trypsin into three major fragments with molecular masses of 27 kDa, 50 kDa, and 20 kDa [lo, 111 which are aligned in this order relative to the NH2-terminus of the heavy chain [12]. Two heavy-chain segments (about ...

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“…Conformational changes within the myosin head during steady-state ATP hydrolysis have been detected by observing changes in the UV absorbance [53], intrinsic fluorescence [30,31,54], biochemical crosslinking [55,56], electron paramagnetic resonance spectroscopy [57][58][59], electron microscopy [60][61][62], NMR spectroscopy [63], fluorescent labeling [64], fluorescence energy transfer [65], small-angle X-ray scattering [66]. In addition, fluorescence polarization is highly sensitive to conformational changes in myosin [67][68][69].…”

Section: Conformational Studies Of Myosin S1mentioning

confidence: 99%

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Gawalapu¹,

Root²

2006

Archives of Biochemistry and Biophysics

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Gawalapu, Ravi Kumar. Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft. Doctor of Philosophy (Biochemistry), August 2007, 148 pp., 3 tables, 60 illustrations, 120 references, 120 titles.In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy.Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actinactivated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin

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Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 NMR

Cited by 51 publications

References 33 publications

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method

Conformational transitions in the myosin head induced by temperature, nucleotide and actin

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

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