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David D Thomas

Publications and source records attributed to David D Thomas.

At least 37 records · Page 2Linked to original sources
PubMed2004

Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.

Calcium↗
PubMed2004

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase.

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.

Alanine↗
PubMed2004

The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model.

Quenched-flow mixing was used to characterize the kinetic behavior of the intermediate reactions of the skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase (SERCA1) at 2 and 21 degrees C. At 2 degrees C, phosphorylation of SR Ca-ATPase with 100 microM ATP labeled one-half of the catalytic sites with a biphasic time dependence [Mahaney, J. E., Froehlich, J. P., and Thomas, D. D. (1995) Biochemistry 34, 4864-4879]. Chasing the phosphoenzyme (EP) with 1.66 mM ADP 10 ms after the start of phosphorylation revealed mostly ADP-insensitive E2P (95% of EP(total)), consistent with its rapid formation from ADP-sensitive E1P. The consecutive relationship of the phosphorylated intermediates predicts a decrease in the proportion of E1P ([E1P]/[EP(total)]) with increasing phosphorylation time. Instead, after 10 ms the proportion of E1P increased and that of E2P decreased until they reached a constant 1:1 stoichiometry ([E1P]:[E2P] approximately 1). At 21 degrees C, phosphorylation displayed a transient overshoot associated with an inorganic phosphate (P(i)) burst, reflecting increased turnover of E2P at the higher temperature. The P(i) burst exceeded the decay of the EP overshoot, suggesting that rephosphorylation of the enzyme occurs before the recycling step (E2 --> E1). This behavior and the reversed order of accumulation of phosphorylated intermediates at 2 degrees C are not compatible with the conventional linear consecutive reaction mechanism: E1 + ATP --> E1.ATP --> E1P + ADP --> E2P --> E2.P(i) --> E1 + P(i). Solubilization of the Ca-ATPase into monomers using the nonionic detergent C(12)E(8) gave a pattern of phosphorylation in which E1P and E2P behave like consecutive intermediates. Kinetic modeling of the C(12)E(8)-solubilized SR Ca-ATPase showed that it behaves according to the conventional Ca-ATPase reaction mechanism, consistent with monomeric catalytic function. We conclude that the nonconforming features of native SERCA1 arise from oligomeric protein conformational interactions that constrain the subunits to a staggered or out-of-phase mode of operation.

Adenosine Diphosphate↗
PubMed2004

Aqueous sample in an EPR cavity: sensitivity considerations.

The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE(011) mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample's dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter Lambda. The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.

Algorithms↗
PubMed2004

(1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles.

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.

Calcium-Binding Proteins↗
PubMed2003

Muscle activity and aging affect myosin structural distribution and force generation in rat fibers.

The purpose of this study was to determine whether increased muscle activity could reverse myosin structural alterations that occur in aged rat muscle and whether those alterations could be induced in young rat muscle by decreased activity. Semimembranosus muscle activity was increased by electrical stimulation (200-ms trains, 154 Hz, 5 V) through a nerve cuff on the tibial branch of the ischiatic nerve. The protocol consisted of 5 sets of 6-10 maximal isometric contractions performed twice per week for 4 or 8-10 wk. Decreased muscle activity was induced by denervation of the semimembranosus muscle for 2 or 4 wk. Semimembranosus fibers were then studied for Ca(2+)-activated force generation. Fibers were also spin labeled on the myosin catalytic domain and studied using electron paramagnetic resonance (EPR) spectroscopy to assess myosin structural distribution. Increased muscle activity for 4 and 8-10 wk in approximately 32-mo-old rats resulted in -16 and +4% changes in specific tension, respectively (P < 0.01). EPR spectra showed that the fraction of myosin heads in the strong-binding structural state during contraction was reduced at 4 wk (0.241 +/- 0.020 vs. 0.269 +/- 0.018, P = 0.046) but returned to normal by 8-10 wk (P = 0.67). Decreased muscle activity for 2 and 4 wk in approximately 9-mo-old rats resulted in 23 and 34% reductions, respectively, in specific tension; EPR spectra showed 16 and 35% decreases in strong-binding myosin (P < 0.01). These data support the hypothesis that changes in muscle activity affect muscle strength, at least in part through alterations in myosin structure and function.

Aging↗
PubMed2003

Transient kinetics and mechanics of myosin's force-generating rotation in muscle: resolution of millisecond rotational transitions in the spin-labeled myosin light-chain domain.

We have used electron paramagnetic resonance (EPR) of spin-labeled scallop muscle, in conjunction with laser flash photolysis of caged ATP, to resolve millisecond rotational transitions of the myosin light-chain domain (LCD) during transient force generation. We previously used EPR to resolve two distinct orientations of the LCD [Baker, J. E., Brust-Mascher, I., Ramachandran, S., LaConte, L. E., and Thomas, D. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2944-2949], correlated these structural states with biochemical states in the actin-myosin ATPase reaction, and showed that a small shift in the steady-state distribution between these two LCD orientations (i.e., a net lever arm rotation) is associated with force generation in muscle. In the study presented here, we measured millisecond changes in this orientational distribution (i.e., the rates of transition between the two LCD orientations) in muscle following flash photolysis of caged ATP, in both the presence and absence of Ca. The transient acquired in the absence of Ca is dominated by a rapid (1/tau(1) = 37 s(-1)) disordering transition from the single orientation in rigor to the bimodal orientation distribution observed for detached cross-bridges in relaxation (i.e., the reversal of the lever arm rotation), followed by a recovery phase (1/tau(2) = 2.4 s(-1)) of very small amplitude (small fraction of heads participating). In the presence of Ca, the transient exhibited a similar initial disordering phase (1/tau(1) = 38.5 s(-1)), followed by a recovery phase (1/tau(2) = 8.33 s(-1)) of substantial amplitude, corresponding to the forward rotation and ordering of the lever arm. A standard kinetic model was used to fit these data, revealing rate constants consistent with those previously determined by other methods. Surprisingly, a comparison of the EPR transients with force transients reveals that the rate of force development (91 s(-1)) is faster than the rate of the forward lever arm rotation (8 s(-1)). This observed relationship between the kinetics of the lever arm rotation and transient force development in muscle provides new insight into how myosin both generates and responds to muscle force.

Actins↗
PubMed2003

Defining the molecular components of calcium transport regulation in a reconstituted membrane system.

Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to determine accurately not only the PLB effects on K(Ca) (Ca concentration at half-maximal activity) of the Ca-ATPase, but also the effects on V(max) (maximal activity). Wild-type PLB (WT-PLB) and two gain-of-function mutants, N27A-PLB and I40A-PLB, showed not only the previously reported increase in K(Ca), but also an increase in V(max). Specifically, V(max) increases linearly with the intramembrane PLB concentration, and is approximately doubled when the sample composition approaches that of cardiac SR. Upon phosphorylation of PLB at Ser-16, the K(Ca) effects were almost completely reversed for WT- and N27A-PLB but were only partially reversed for I40A-PLB. Phosphorylation induced a V(max) increase for WT-PLB, and a V(max) decrease for N27A- and I40A-PLB. We conclude that PLB and PLB phosphorylation affect V(max) as well as K(Ca), and that the magnitude of both effects is sensitive to the PLB concentration in the membrane.

Calcium↗
PubMed2003

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1.

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.

Animals↗
PubMed2003

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban.

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.

Buffers↗
PubMed2003

NMR solution structure and topological orientation of monomeric phospholamban in dodecylphosphocholine micelles.

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.

Amino Acid Sequence↗
PubMed2002

Coordination of the two heads of myosin during muscle contraction.

We have used luminescence resonance energy transfer between regulatory light chains (RLC) to detect structural changes within the dimeric myosin molecule in contracting muscle fibers. Fully functional scallop muscle fibers were prepared such that each myosin molecule contained a terbium-labeled (luminescent donor) RLC on one head and a rhodamine-labeled (acceptor) RLC on the other. Time-resolved luminescence energy transfer between the two heads increased upon the transition from relaxation (ATP) to contraction (ATP plus Ca) and increased further in rigor (no ATP). Combined with experiments on mutant RLCs labeled specifically at other sites, these results support a model in which the force-generating weak-to-strong transition causes one myosin LC domain to tilt through a 30 degrees angle toward the other, thus acting as a coordinated lever arm.

Animals↗
PubMed2002

Interprotein electron transfer in a confined space: uncoupling protein dynamics from electron transfer by sol-gel encapsulation.

In this paper, we describe the first observations of photoinitiated interprotein electron transfer (ET) within sol-gels. We have encapsulated three protein-protein complexes, specifically selected because they represent a full range of affinities, are sensitive to different types of dynamic processes, and thus are expected to respond differently to sol-gel encapsulation. The three systems are (i) the [Zn, Fe(3+)L] mixed-metal hemoglobin hybrids, where the alpha(1)-Zn and beta(2)-Fe subunits correspond to a "predocked" protein-protein complex with a crystallographically defined interface (Natan, M. J.; Baxter, W. W.; Kuila, D.; Gingrich, D. J.; Martin, G. S.; Hoffman, B. M. Adv. Chem. Ser. 1991, 228 (Electron-Transfer Inorg., Org., Biol. Syst.), 201-213), (ii) the Zn-cytochrome c peroxidase complex with cytochrome c, [ZnCcP, Fe(3+)Cc], having an intermediate affinity between its partners (Nocek, J. M.; Zhou, J. S.; De Forest, S.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. Rev. 1996, 96, 2459-2489), and (iii) the [Zn-deuteromyoglobin, ferricytochrome b(5)] complex, [ZnDMb, Fe(3+)b(5)], which is loosely bound and highly dynamic (Liang, Z.-X.; Nocek, J.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.; Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849-6859. Intersubunit ET within the hybrid does not involve second-order processes or subunit rearrangements, and thus is influenced only by perturbations of high-frequency motions coupled to ET. For the latter two complexes, sol-gel encapsulation eliminates second-order processes: protein partners encapsulated as a complex must stay together throughout a photoinitiated ET cycle, while proteins encapsulated alone cannot acquire a partner. It further modulates intracomplex motions of the two partners.

Crystallography↗
PubMed2002

Structure and orientation of sarcolipin in lipid environments.

Sarcolipin (SLN) is a 31 amino acid integral membrane protein that regulates Ca-ATPase activity in skeletal muscle. Here, we report the three-dimensional structure and topology of synthetic SLN in lipid environments, as determined by solution and solid-state NMR spectroscopy. 2D solution NMR experiments were performed on SLN solubilized in sodium dodecyl sulfate (SDS) micelles. We found that SLN adopts a highly defined alpha-helical conformation from F9 through R27, with a backbone RMSD of 0.65 A and a side chain RMSD of 1.66 A. The N-terminus (M1 through L8) and the C-terminus (S28 through Y31) are mostly unstructured. The orientation of the SLN was determined using one-dimensional (15)N NMR solid-state spectroscopy. The protein was incorporated into phospholipid bilayers prepared from a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. The (15)N chemical shift solid-state spectra from selectively labeled SLN samples indicate that SLN orients perpendicularly to the plane of the membrane bilayers. These results support the proposed mechanism of Ca-ATPase regulation of SLN via protein-protein intramembranous interactions between the highly conserved transmembrane domains of the Ca-ATPase and the conserved transmembrane domain of SLN.

Amino Acid Sequence↗
PubMed2002

Changes in actin and myosin structural dynamics due to their weak and strong interactions.

Figure 3 summarizes the effects of actomyosin binding on the internal and global dynamics of either protein, as discussed in this chapter. These effects depend primarily on the strength of the interaction; which in turn depends on the state of the nucleotide at the myosin active site. When either no nucleotide or ADP is bound, the interaction is strong and the effect on each protein is maximal. When the nucleotide is ATP or ADP.Pi, or the equivalent nonhydrolyzable analogs, the interaction is weak and the effect on molecular dynamics of each protein is minimal. The weaker effects in weak-binding states are not simply the reflection of lower occupancy of binding sites--the molecular models in Fig. 3 illustrate the effects of the formation of the ternary complex, after correction for the free actin and myosin in the system. Thus EPR on myosin (Berger and Thomas 1991; Thomas et al. 1995) and pyrene fluorescence studies on actin (Geeves 1991) have shown that the formation of a ternary complex has a negligible effect on the internal dynamics of both [figure: see text] proteins (left side of Fig. 3, white arrows). As shown by both EPR (Baker et al. 1998; Roopnarine et al. 1998) and phosphorescence (Ramachandran and Thomas 1999), both domains of myosin are dynamically disordered in weak-binding states, and this is essentially unaffected by the formation of the ternary complex (left side of Fig. 3, indicated by disordered myosin domains). The only substantial effect of the formation of the weak interaction that has been reported is the EPR-detected (Ostap and Thomas 1991) restriction of the global dynamics of actin upon weak myosin binding (left column of Fig. 3, gray arrow). The effects of strong actomyosin formation are much more dramatic. While substantial rotational dynamics, both internal and global, exist in both myosin and actin in the presence of ADP or the absence of nucleotides, spin label EPR, pyrene fluorescence, and phosphorescence all show dramatic restrictions in these motions upon formation of the strong ternary complex (right column of Fig. 3). One implication of this is that the weak-to-strong transition is accompanied by a disorder-to-order transition in both actin and myosin, and this is itself an excellent candidate for the structural change that produces force (Thomas et al. 1995). Another clear implication is that the crystal structures obtained for isolated myosin and actin are not likely to be reliable representations of structures that exist in ternary complexes of these proteins (Rayment et al. 1993a and 1993b; Dominguez et al. 1998; Houdusse et al. 1999). This is clearly true of the strong-binding states, since the spectroscopic studies indicate consistently that substantial changes occur in both proteins upon strong complex formation. For the weak complexes, the problem is not that complex formation induces large structural changes, but that the structures themselves are dynamically disordered. This is probably why so many different structures have been obtained for myosin S1 with nucleotides bound--each crystal is selecting one of the many different substates represented by the dynamic ensemble. Finally, there is the problem that the structures of actomyosin complexes are probably influenced strongly by their mechanical coupling to muscle protein lattice (Baker at al. 2000). Thus, even if co-crystals of actin and myosin are obtained in the future, an accurate description of the structural changes involved in force generation will require further experiments using site-directed spectroscopic probes of both actin and myosin, in order to detect the structural dynamics of these ternary complexes under physiological conditions.

Actins↗
PubMed2002

Molecular dynamics simulation of site-directed spin labeling: experimental validation in muscle fibers.

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.

Animals↗
PubMed2002

D-dimer and calf circumference in the evaluation of outpatient deep venous thrombosis.

OBJECTIVE: Duplex ultrasonography (DU) is the primary method for diagnosis of deep venous thrombosis (DVT) but is relatively expensive and not always readily available. Attempts to exclude the diagnosis of DVT with D-dimer or clinical criteria independently have been unsuccessful. The goal of our study was to evaluate a second-generation rapid quantitative D-dimer and simple clinical parameters for screening of outpatients for DVT. PATIENTS AND METHODS: Patients undergoing DU of the lower extremities for suspected DVT were prospectively evaluated. Patients undergoing lower extremity venous ultrasound scan for suspected pulmonary embolism or already on anticoagulant therapy were excluded from the study. Data were analyzed to assess the optimal combination of characteristics to include and exclude proximal DVT. RESULTS: One hundred fifty-six outpatients met inclusion criteria and were enrolled in the study. Elevated levels of D-dimer of 0.5 ng/mL or more were noted in 21 of 22 patients diagnosed with DVT, yielding a sensitivity of 95% and negative predictive value of 99%. Subjective symptoms of swelling or pain were present in 94% of all outpatients. Asymmetric calf swelling of more than 2.0 cm was noted in 14 of 22 patients (64%) with proximal DVT compared with 22 of 134 patients (16%) without DVT (P <.003). No single clinical history variable was significant on multivariate analysis. All outpatients with proximal DVT had either leg swelling of more than 2 cm or a positive D-dimer. CONCLUSION: A combination of a second-generation quantitative D-dimer and calf measurement provides an easy and effective means of excluding proximal DVT when screening outpatients. Patients with calf circumference 2.0 cm or less and a negative D-dimer may undergo nonemergent DU. Patients with a positive D-dimer or asymmetrical calf swelling of more than 2.0 cm alone or in combination should undergo emergent DU.

Female↗