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Abstract Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A 15 G 15 ) in aqueous solutions containing 5mM MgCl 2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A 15 ) from that of d(A 15 G 15 ), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10°C and 60°C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T 15 ) to the solution, complexes with the adenine portion of the d(A 15 G 15 ) frayed wires are formed. Subtraction of d(A 15 ):d(T 15 ) spectra measured at several temperatures from those of the d(A 15 G 15 ):d(T 15 ) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A 15 ):d(T 15 ) duplex is identical to that of the unbinding of d(T 15 ) from d(A 15 G 15 ):d(T 15 ) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide.

Measurements of the proton second and fourth moments have been undertaken for multilamellar dispersions, in excess water, of protiated and chain-perdeuterated dipalmitoyl phosphatidylcholine (DPPC) in the temperature range -20 to 50 degrees C. The comparison of the measured moment with the rigid lattice M2 and the calculated second moment values in the presence of certain motions gives insight into the dynamic structure of the methylene chains of DPPC. This study demonstrates that at -15 degrees C there is still a significant amount of methylene chain motion or disorder in DPPC. At 35 degrees C the moment values indicate that the methylene chains are not in the fully extended all-trans conformation and they may also be rotationally disordered. At the pretransition there is a decrease in magnitude of the proton second moments, which can be accounted for by an increase in the lateral diffusion rate to a value greater than 10(-11) cm2/s. This work suggests that at temperatures just below the main transition the chain conformation is considerably different from the common model structure (P beta') in which the chains are fully extended. In the liquid-crystalline phase the proton moment data are in agreement with data from other techniques on the liquid-like nature of the methylene chains. It is demonstrated how the values of the moment ratio M4r/(M2r)2, which are relatively constant within each phase, can be used to calculate the molar fractions of coexisting liquid-crystalline and gel-phase phospholipid at temperatures near the main transition.

The structural phase behavior of phospholipid mixtures consisting of short-chain (dihexanoyl phosphatidylcholine) and long-chain lipids (dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol), with and without lanthanide ions was investigated by small-angle neutron scattering (SANS). SANS profiles were obtained from 10 degrees C to 55 degrees C using lipid concentrations ranging from 0.0025 g/ml to 0.25 g/ml. The results reveal a wealth of distinct morphologies, including lamellae, multi-lamellar vesicles, unilamellar vesicles, and bicellar disks.

Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both phospholamban and sarcolipin naturally associate with SERCA.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.

Kv1.2 is a prominent neuronal potassium channel subtype linked to severe epilepsies and movement disorders. We have demonstrated that neuronal and heterologously-expressed Kv1.2 channels exhibit use-dependent activation, a behavior characterized by progressive potentiation of channel activity during trains of repetitive stimuli. Use-dependent activation arises from a gating mode shift from a ‘reluctant’ slowly-activating mode to a ‘willing’ rapidly-activating mode with hyperpolarized voltage-dependence of activation. Moreover, Kv1.2 subunits recruit this use-dependent phenotype to heteromeric channel complexes. In this study, we demonstrate that use-dependent activation of Kv1.2 channel complexes is strongly regulated by a variety of exogenous and physiological redox species. Under ambient redox conditions, Kv1.2 channels exhibit marked cell-to-cell variability of use-dependent activation. However, exposure to mild reducing conditions normalizes this response, such that a pronounced use-dependent phenotype is consistently observed, together with a dramatic depolarizing shift of voltage-dependent activation with a V1/2 of 51+/- 6 mV. Mutagenesis of candidate cysteine residues in Kv1.2 did not affect redox sensitivity, therefore we hypothesize a role for an extrinsic redox-sensitive interacting partner. Using a variety of redox buffers, we demonstrate that use-dependent activation is steeply regulated by redox potentials between −50 and −100 mV, within the typical extracellular range. Furthermore, effects of membrane-impermeable reducing agents demonstrate that use-dependent activation is regulated by the extracellular redox state. Taken together, these findings suggest that Kv1.2 is a unique transducer of the extracellular environment, and may translate altered extracellular redox conditions to changes in cellular electrical function.

Voltage sensing in Kv channels originates from the coupling of movement between the charged S4 segment and the activation gate at the cytoplasmic region of the pore domain. The voltage sensor moves prior to the pore opening and the pore must shut before the voltage sensor returns to its resting state. However, gating current recordings from Kv channels indicate that frequently voltage sensors return more slowly after depolarisations that populate open states, indicating that the open pore exerts a resistance to S4 return. This process of pore closure therefore intrinsically regulates the deactivation kinetics of Kv channels. We observed slow voltage sensor return (IgOFF) in WT-Kv1.2 channels under non-permeant ionic conditions after depolarisations to voltages that caused channel openings. Using TEA+ and NMG+ internal solutions resulted in a slower IgOFF than internal Cs+, suggesting that the intracellular ionic composition was modulating IgOFF. A mutation in the pore lining S6 segment to enlarge the inner cavity (Kv1.2-I402C) removed the slowing of IgOFF in the presence of internal NMG+, suggesting that NMG+ interacted within the inner cavity of the WT channel to prevent pore closure through a ‘foot in the door’ mechanism. Gating currents of a non-conducting, P-type inactivated channel (Kv1.2-W366F, V381T), in the presence of intracellular K+ ions also displayed a slowing of IgOFF after depolarisations that would open the channel pore. These results suggest that internal K+ ions bound in the inner cavity can also slow activation gate closure. We propose that internal ions in the cavity of Kv1.2 allosterically regulate the voltage sensor deactivation kinetics by preventing pore closure and thus rate limiting the return of voltage sensors.

Calcins is a novel family of scorpion peptides that bind with high affinity and specificity to ryanodine receptors (RyRs) and increase their open probability by inducing the appearance of a long-lasting subconductance state. Here we report two newly-identified calcins (Vejocalcin and Urocalcin) and provide a comprehensive analysis of the structure-function relationship of the eight calcins known to date, based on primary sequence examination, 3D structure modeling, and their effect on RyR1. [3H]Ryanodine binding assays, used as index of the open probability (Po) of RyRs, revealed that all eight calcins (Opicalcin1, Opicalcin2, Imperacalcin, Maurocalcin, Hadrucalcin, Hemicalcin, Vejocalcin, and Urocalcin) activate RyR1 dose-dependently, with EC50s (in nM) 0.35, 5, 8, 12, 37, 71, 91, and 523, respectively. At 1 µM, calcins significantly augmented the bell-shaped [Ca2+]-activity curve of RyR1 with potency Opicalcin1>Opicalcin2>Vejocalcin>Urocalcin. In single channel recordings, the heretofore uncharacterized calcins Opicalcin1, Opicalcin2 and Vejocalcin increased the Po of RyR1 significantly and the fractional conductance was ∼0.45, 0.30, and 0.65, respectively, of the full conductance state. Opicalcin1, Maurocalcin, Hadrucalcin and Vejocalcin induced Ca2+ release from rabbit skeletal SR with ED50s (in nM) 2.8±0.02, 8.3±0.25, 16±0.4, 36±0.5, respectively. A clear Ca2+ release-[3H]ryanodine binding activity correlation (r2=0.99) was obtained. Primary sequence alignment and evolutionary analysis (ClustalW and MEGA5.2) showed high homology among all calcins. An inhibitor cysteine-knot (ICK) motif with βαββ domains stabilized by three disulfide bridges characterizes this peptide family, although Vejocalcin lacks the β2 sheet. Positively-charged residue mutations in the high variable N-terminal region (G1-N14) and negatively-charged residue variations in the conserved C-terminal region (D15-R33) greatly decrease the effect of calcins on RyR1. In conclusion, natural variation in calcin peptides offers a diversified set of RyR ligands with capacity to modulate RyRs with high dynamic range and potency.