Search

The type 2a sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2a) plays a central role in the intracellular Ca 2+ homeostasis of cardiac myocytes, pumping Ca 2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca 2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca 2+ -ATPase and Ca 2+ transport activities in cardiac SR vesicles, and on Ca 2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca 2+ -ATPase and Ca 2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca 2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca 2+ load by enhancing SERCA2a-mediated Ca 2+ transport. Analysis of cytosolic Ca 2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca 2+ transients and SR Ca 2+ load, with negligible effects on L-type Ca 2+ channels and Na + /Ca 2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca 2+ transients and SR Ca 2+ load, but it decreased the propensity toward Ca 2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [ 3 H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca 2+ and a small increase in μM Ca 2+ . Accordingly, compound 1 slightly decreased Ca 2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca 2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca 2+ uptake, as found in failing hearts., Competing Interests: Declaration of interests R.L.C. and D.D.T. hold equity in, and serve as executive officers for, Photonic Pharma LLC, which had no role in this study. These relationships have been reviewed and managed by the University of Minnesota., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca 2+ , the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca 2+ , the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca 2+ , the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca 2+ stabilized the closed conformation by a factor of two. We conclude that the Ca 2+ -dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca 2+ -dependent structural dynamics of bound CaM., (Published by Elsevier Inc.)

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible., (Copyright © 2019. Published by Elsevier Inc.)

Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women. Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma. Here we show that AR and EGR1 bind to the long non-coding RNA SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes. ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites. We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21 Waf1/Cip1 . Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1's intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr 112 disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have used chemical synthesis, electron paramagnetic resonance (EPR), and circular dichroism to detect and analyze the structural dynamics of a ryanodine receptor (RyR) peptide bound to calmodulin (CaM). The skeletal muscle calcium release channel RyR1 is activated by Ca 2+ -free CaM and inhibited by Ca 2+ -bound CaM. To probe the structural mechanism for this regulation, wild-type RyRp and four spin-labeled derivatives were synthesized, each containing the nitroxide probe 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid substituted for a single amino acid. In 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid, the probe is rigidly and stereospecifically coupled to the α-carbon, enabling direct detection by EPR of peptide backbone structural dynamics. In the absence of CaM, circular dichroism indicates a complete lack of secondary structure, while 40% trifluoroethanol (TFE) induces >90% helicity and is unperturbed by the spin label. The EPR spectrum of each spin-labeled peptide indicates nanosecond dynamic disorder that is substantially reduced by TFE, but a significant gradient in dynamics is observed, decreasing from N- to C-terminus, both in the presence and absence of TFE. When bound to CaM, the probe nearest RyRp's N-terminus shows rapid rotational motion consistent with peptide backbone dynamics of a locally unfolded peptide, while the other three sites show substantial restriction of dynamics, consistent with helical folding. The two N-terminal sites, which bind to the C-lobe of CaM, do not show a significant Ca 2+ -dependence in mobility, while both C-terminal sites, which bind to the N-lobe of CaM, are significantly less mobile in the presence of bound Ca 2+ . These results support a model in which the interaction of RyR with CaM is nonuniform along the peptide, and the primary effect of Ca 2+ is to increase the interaction of the C-terminal portion of the peptide with the N-terminal lobe of CaM. These results provide, to our knowledge, new insight into the Ca 2+ -dependent regulation of RyR by CaM., (Copyright © 2016. Published by Elsevier Inc.)

We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K(+) and 3 mM Mg(2+). Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K(+) ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K(+) in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H(+)771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca(2+)-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser(16). To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser(16) phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M'.D.P, we used pPDM to cross-link SH1 (Cys(707)) to SH2 (Cys(697)) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M'.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M'D.P analog on the force generation pathway., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In a previous study, sarcolipin (SLN) was shown to form channels selective toward chloride ion when incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM). Its incorporation had only a modest permeabilizing effect on phosphate ion. In this note the resistance of a tBLM membrane incorporating sarcolipin was investigated by electrochemical impedance spectroscopy in aqueous solutions of 0.05 M sodium phosphate of pH ranging from 5.3 to 8, in the presence of ATP, adenosine monophosphate, and phenylphosphonic acid. At pH 5.3, submicromolar additions of ATP increase the conductivity of the tBLM incorporating SLN up to a maximum limiting value. The dependence of the conductivity on the ATP concentration satisfies the Michaelis-Menten equation, with an association constant of 0.1 microM. Phenylphosphonium ion and adenosine monophosphate exert an inhibitory effect on membrane permeabilization to phosphate ions by ATP if they are added before ATP, but not if they are added after it. An explanation for this behavior is provided. In conclusion, SLN acts as an ATP-induced phosphate carrier exhibiting a behavior quite similar to that of the unidentified P(i) transporter described previously. No ion-channel activity is exhibited by the T18A mutant of SLN.

We have used a bifunctional spin label (BSL) to cross-link Cys(707) (SH1) and Cys(697) (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A.M.ADP.P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A.M.ADP. In contrast, helical order is observed for the strongly bound state A.M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A.M.ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A.M.ADP, slow rotational mobility comparable to that of A.M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

Sarcolipin was incorporated into a lipid bilayer anchored to a mercury electrode through a hydrophilic tetraethyleneoxy chain. The behavior of this tethered bilayer lipid membrane incorporating sarcolipin was investigated by electrochemical impedance spectroscopy and by recording charge versus time curves after potential jumps. When the transmembrane potential starts to become negative on the trans side, evidence is provided that sarcolipin aggregates into ion-conducting pores. Over the range of physiological transmembrane potentials, these pores are permeable to small inorganic anions such as chloride, phosphate, or sulfate but impermeable to inorganic cations such as Na+ and K+. Only at transmembrane potentials more negative than approximately -150 mV on the trans side do sarcolipin channels allow the translocation of the latter cations. A tentative relationship between this property of sarcolipin and its regulatory function on Ca-ATPase of sarcoplasmic reticulum is proposed.

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.

We have performed molecular dynamics simulations of the phosphorylated (at S-19) and the unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin to provide insight into the structural basis of regulation. This domain does not appear in any crystal structure, so these simulations were combined with site-directed spin labeling to define its structure and dynamics. Simulations were carried out in explicit water at 310 K, starting with an ideal alpha-helix. In the absence of phosphorylation, large portions of the domain (residues S-2 to K-11 and R-16 through Y-21) were metastable throughout the simulation, undergoing rapid transitions among alpha-helix, pi-helix, and turn, whereas residues K-12 to Q-15 remained highly disordered, displaying a turn motif from 1 to 22.5 ns and a random coil pattern from 22.5 to 50 ns. Phosphorylation increased alpha-helical order dramatically in residues K-11 to A-17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S-19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the root mean-square deviation. These results on the isolated phosphorylation domain, predicting a disorder-to-order transition induced by phosphorylation, are remarkably consistent with published experimental data involving site-directed spin labeling of the intact RLC bound to the two-headed heavy meromyosin. The simulations provide new insight into structural details not revealed by experiment, allowing us to propose a refined model for the mechanism by which phosphorylation affects the N-terminal domain of the RLC of smooth muscle myosin.

We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban (PLB), a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300 K for three peptides spanning the first 25 residues of PLB: wild-type (PLB(1-25)), PLB(1-25) phosphorylated at Ser16 and PLB(1-25) with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26-ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11-16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11-21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3-11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser-16 and Arg-13. Replacing Arg-9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser-16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.

We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an "L"-shaped structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees (Zamoon et al., 2003; Mascioni et al., 2002). T(1), T(2), and (1)H/(15)N nuclear Overhauser effect values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22-30). In addition, using Carr-Purcell-Meiboom-Gill-based experiments, we have characterized phospholamban dynamics in the micros-ms timescale. We found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the micros-ms range, whereas minimal dynamics were detected for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and the cytoplasmic helix. We propose that these dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.

Phospholamban is an integral membrane protein that regulates the contractility of cardiac muscle by maintaining cardiomyocyte calcium homeostasis. Abnormalities in association of protein kinase A with PLB have recently been linked to human heart failure, where a single mutation is responsible for dilated cardiomyopathy. To date, a high-resolution structure of phospholamban in a lipid environment has been elusive. Here, we describe the first structure of recombinant, monomeric, biologically active phospholamban in lipid-mimicking dodecylphosphocholine micelles as determined by multidimensional NMR experiments. The overall structure of phospholamban is "L-shaped" with the hydrophobic domain approximately perpendicular to the cytoplasmic portion. This is in agreement with our previously published solid-state NMR data. In addition, there are two striking discrepancies between our structure and those reported previously for synthetic phospholamban in organic solvents: a), in our structure, the orientation of the cytoplasmic helix is consistent with the amphipathic nature of these residues; and b), within the hydrophobic helix, residues are positioned on two discrete faces of the helix as consistent with their functional roles ascribed by mutagenesis. This topology renders the two phosphorylation sites, Ser-16 and Thr-17, more accessible to kinases.

We have developed a computational molecular dynamics technique to simulate the motions of spin labels bound to the regulatory domain of scallop myosin. These calculations were then directly compared with site-directed spin labeling experimental results obtained by preparing seven single-cysteine mutants of the smooth muscle (chicken gizzard) myosin regulatory light chain and performing electron paramagnetic resonance experiments on these spin-labeled regulatory light chains in functional scallop muscle fibers. We determined molecular dynamics simulation conditions necessary for obtaining a convergent orientational trajectory of the spin label, and from these trajectories we then calculated correlation times, orientational distributions, and order parameters. Simulated order parameters closely match those determined experimentally, validating our molecular dynamics modeling technique, and demonstrating our ability to predict preferred sites for labeling by computer simulation. In several cases, more than one rotational mode was observed within the 14-ns trajectory, suggesting that the spin label samples several local energy minima. This study uses molecular dynamics simulations of an experimental system to explore and enhance the site-directed spin labeling technique.

Dystrophin has been shown to be associated in cells with actin bundles. Dys-246, an N-terminal recombinant protein encoding the first 246 residues of dystrophin, includes two calponin-homology (CH) domains, and is similar to a large class of F-actin cross-linking proteins including alpha-actinin, fimbrin, and spectrin. It has been shown that expression or microinjection of amino-terminal fragments of dystrophin or the closely related utrophin resulted in the localization of these protein domains to actin bundles. However, in vitro studies have failed to detect any bundling of actin by either intact dystrophin or Dys-246. We show here that the structure of F-actin can be modulated so that there are two modes of Dys-246 binding, from bundling actin filaments to only binding to single filaments. The changes in F-actin structure that allow Dys-246 to bundle filaments are induced by covalent modification of Cys-374, proteolytic cleavage of F-actin's C-terminus, mutation of yeast actin's N-terminus, and different buffers. The present results suggest that F-actin's structural state can have a large influence on the nature of actin's interaction with other proteins, and these different states need to be considered when conducting in vitro assays.

If, contrary to conventional models of muscle, it is assumed that molecular forces equilibrate among rather than within molecular motors, an equation of state and an expression for energy output can be obtained for a near-equilibrium, coworking ensemble of molecular motors. These equations predict clear, testable relationships between motor structure, motor biochemistry, and ensemble motor function, and we discuss these relationships in the context of various experimental studies. In this model, net work by molecular motors is performed with the relaxation of a near-equilibrium intermediate step in a motor-catalyzed reaction. The free energy available for work is localized to this step, and the rate at which this free energy is transferred to work is accelerated by the free energy of a motor-catalyzed reaction. This thermodynamic model implicitly deals with a motile cell system as a dynamic network (not a rigid lattice) of molecular motors within which the mechanochemistry of one motor influences and is influenced by the mechanochemistry of other motors in the ensemble.

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force? Pate and Cooke (1989. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.

We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 microM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 microM) than at high (10 microM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.

We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.

We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the microsecond time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45 degree transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (microsecond) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction,indicating that the light-chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.

We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or 0.5 M lithium chloride protected against lidocaine-induced inhibition, indicating that electrostatic interactions are essential to lidocaine inhibition of the Ca-ATPase. The phospholamban (PLB) antibody 2D12, which mimics PLB phosphorylation, had no effect on lidocaine inhibition of the Ca-ATPase in cardiac SR. Inhibition and aggregation of the Ca-ATPase in cardiac SR occurred at lower concentrations of lidocaine than necessary to inhibit and aggregate the Ca-ATPase in skeletal SR, suggesting that the cardiac isoform of the enzyme has a higher affinity for lidocaine. Halothane inhibited and aggregated the Ca-ATPase in cardiac SR. Both inhibition and aggregation of the Ca-ATPase by halothane were much greater in the presence of PLB antibody or when PLB was phosphorylated, indicating a protective effect of PLB on halothane-induced inhibition and aggregation. The effects of halothane on cardiac SR are opposite from the effects of halothane observed in skeletal SR, where halothane activates and dissociates the Ca-ATPase. These results underscore the crucial role of protein-protein interactions on Ca-ATPase regulation and anesthetic perturbation of cardiac SR.

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme. When combined with the effects of other inhibitors on the Ca-ATPase, these results support a general model for the coupling of enzyme conformation and self-association in this system.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.

The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban increases the rotational mobility of the Ca-ATPase in the sarcoplasmic reticulum bilayer, due to a decrease in large-scale protein association, with a [Ca2+] dependence parallel to that of enzyme activation. These results support a model in which phospholamban phosphorylation or calcium free the enzyme from a kinetically unfavorable associated state.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.