Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1.

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.

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.

Mechanism of Ca-ATPase inhibition by melittin in skeletal sarcoplasmic reticulum.

We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and steady-state fluorescence experiments in combination with enzyme kinetic assays, utilizing (1) native and charge-modified melittin in order to characterize the peptide charge dependence of the melittin-SR interaction, and (2) various calcium levels in order to define the effect of melittin on the enzyme's E1 and E2 conformational equilibrium. TPA results showed that decreasing melittin's positive charge dramatically decreases the ability of the peptide to aggregate the enzyme, which correlates with a reduced potency of the modified peptide to inhibit enzymatic activity. Steady-state fluorescence of fluorescein isothiocyanate-labeled Ca-ATPase showed that melittin reduces Ca-ATPase affinity for calcium by shifting the enzyme's E1-E2 conformational equilibrium toward E2, but increasing calcium progressively reverses this shift. Kinetic experiments showed that melittin does not prevent ATP-dependent enzyme phosphorylation, but it completely inhibits Pi-dependent EP formation and substantially slows Pi release during steady-state cycling. We conclude that melittin-induced aggregation of the Ca-ATPase depends on the electrostatic interaction of the peptide with cytoplasmic Ca(2+)-dependent sites on the enzyme, and that enforced Ca-ATPase protein-protein interactions inhibit the conformational transitions that facilitate phosphoenzyme hydrolysis.

Protein and lipid rotational dynamics in cardiac and skeletal sarcoplasmic reticulum detected by EPR and phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy to detect the rotational dynamics of the Ca-ATPase and its associated lipids in dog cardiac sarcoplasmic reticulum (DCSR), in comparison with rabbit skeletal SR (RSSR), in order to obtain insight into the physical bases for different activities and regulation in the two systems. Protein rotational motions were studied with time-resolved phosphorescence anisotropy (TPA) of erythrosin isothiocyanate (ERITC) and saturation-transfer EPR (ST-EPR) of a maleimide spin-label (MSL). Both labels were attached selectively and rigidly to the Ca-ATPase. Lipid rotational motions were studied with conventional EPR of stearic acid spin-labels. As in previous studies on RSSR, the phosphorescence anisotropy decays of both preparations at 4 degrees C were multiexponential, due to the presence of different oligomeric species. The rotational correlation times for the different rotating species were similar for the two preparations, but the total decay amplitude was substantially less for cardiac SR, indicating that more of the Ca-ATPase molecules are in large aggregates in DCSR. ST-EPR spectra confirmed that the Ca-ATPase is less rotationally mobile in DCSR than in RSSR. Lipid probe mobility and fatty acid composition were very similar in the two preparations, indicating that the large differences observed in protein mobility are not due to differences in lipid fluidity. We conclude that the higher restriction in protein mobility observed by both ST-EPR and TPA is due to more extensive protein-protein interactions in DCSR than in RSSR.