Folding and function of the troponin tail domain. Effects of cardiomyopathic troponin T mutations

Troponin contains a globular Ca(2+)-binding domain and an elongated tail domain composed of the N terminus of subunit troponin T (TnT). The tail domain anchors troponin to tropomyosin and actin, modulates myosin function, and is a site of cardiomyopathy-inducing mutations. Critical interactions between tropomyosin and troponin are proposed to depend on tail domain residues 112-136, which are highly conserved across phyla. Most cardiomyopathy mutations in TnT flank this region. Three such mutations were examined and had contrasting effects on peptide TnT-(1-156), promoting folding and thermal stability assessed by circular dichroism (F110I) or weakening folding and stability (T104V and to a small extent R92Q). Folding of both TnT-(1-156) and whole troponin was promoted by replacing bovine TnT Thr-104 with human TnT Ala-104, further indicating the importance of this cardiomyopathy site residue for protein folding. Mutation F110I markedly stabilized the troponin tail but weakened binding of holo-troponin to actin-tropomyosin 8-fold, suggesting that loss of flexibility impairs troponin tail function. The effect of the F110I mutation on troponin-tropomyosin binding to actin was much less, indicating this flexibility is particularly important for the interactions of troponin with tropomyosin. We suggest that most cardiomyopathic mutations in the troponin tail alter muscle function indirectly, by perturbing interactions between troponin and tropomyosin requisite for the complex effects of these proteins on myosin.     

muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties.

We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Synthesis, biophysical, and biochemical properties of PNA-DNA chimeras

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T20-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA20 and their hydrolyses by 3'- and 5'-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

Effects of the state of the succinimido-ring on the fluorescence and structural properties of pyrene maleimide-labeled alpha alpha-tropomyosin.

Rabbit skeletal alpha alpha-tropomyosin was labeled at Cys-190 with pyrene maleimide to form (S-[N-(1-pyrene)succinimido])2-tropomyosin (pyreneI-Tm). The product with cleaved succinimido-rings, pyreneII-Tm was also prepared by incubation of pyreneI-Tm at pH greater than 7.5. The pH dependence of the rate of cleavage indicated that hydrolysis rather than aminolysis was the more likely reaction. PyreneI-Tm exhibited a loss in helix content and end-to-end polymerization compared with unlabeled Tm, which increased upon formation of pyreneII-Tm. The cleavage resulted in increased interchain excited state excimer fluorescence originating from pyrene-pyrene interaction between the chains. Thus, increased pyrene-pyrene interaction at Cys 190 leads to an increase in unfolding, the effects of which appear to be transmitted to the ends of tropomyosin. The fluorescence properties of the two types of pyrene-succinimide adducts of dithiothreitol were very similar to the corresponding adducts of pyrene-Tm indicating excimer formation through ground state pyrene-pyrene interaction.     

Sterilization and preservation influence the biophysical properties of human amnion grafts.

The introduction of amniotic membrane (AM) transplantation in ophthalmic surgery holds great promise and in many clinical situations it offers an alternative to existing management options. The purpose of this study was to examine the influence of established sterilization and preservation procedures on biophysical and histological properties of AM grafts. Amnion was sterilized by peracetic acid/ethanol sterilization [PES] and preserved by air-drying (sterile laminar flow) [AD] or in glycerol [GLYC]. Unsterilized AM were preserved at -80 degrees C [-80 degrees C] and served as an experimental control. Amnion allografts were characterized by the determination of their thickness, moisture vapour permeability (MVP), oxygen permeability (OPERM), tensile strength and sulphur content. Immunostaining for tissue-specific and basement membrane-related proteins was performed. Differences in biophysical parameters were found between the unsterilized allografts and the sterilized, air-dried or glycerol-preserved allografts. [PES/AD] showed the highest MVP and OPERM, the highest tensile strength and the lowest sulphur content and thickness. [PES/GLYC] exhibited the lowest OPERM and the highest thickness compared to [-80 degrees C] and [PES/AD]. Collagen types V and VII were preserved the best in the control group. Sterilization and preservation affect biophysical properties important for the use of AM as allogenic grafts. It has to be determined if any change, as noted, has a clinical impact.     

Some biophysical and biochemical properties of poly(phthaloyl L-lysine) microcapsules containing hemolysate.

Measurements of oxygen equilibrium, zeta-potential, resistance to flow, carbonic anhydrase activity, and catalase activity were made on sheep erythrocyte hemolysate-loaded poly(phthaloyl L-lysine) microcapsules (artificial red blood cells) prepared by an interfacial polycondensation technique. The measurements revealed that oxygen dissociation equilibrium, zeta-potential, and carbonic anhydrase activity of the microcapsules are almost the same as those of sheep erythrocytes, while the microcapsules have a higher resistance to flow and a lower catalase activity than the erythrocytes. Possible ways of improving the properties of the microcapsules were suggested.     

Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.     

Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in alpha-tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.     

Synthesis and biophysical properties of polymerized human serum albumin

The use of many plasma expanders (PEs) is often limited by undesirable side effects, such as red blood cell (RBC) aggregation (hydroxyethyl starch), nephrotoxicity (dextran), and extravasation (albumin). Despite its natural prevalence in the bloodstream, human serum albumin (HSA) can increase the risk of mortality when administered to patients with increased vascular permeability (i.e., patients suffering from burns, septic shock, and endothelial dysfunction). The harmful extravasation of HSA can be limited by polymerizing HSA to increase its molecular size. In this study, HSA was nonspecifically cross-linked with glutaraldehyde at different cross-link densities by varying the molar ratio of glutaraldehyde to HSA. The results of this study show that the weight-averaged molecular weight (MW), viscosity, and extent of RBC aggregation of polymerized HSA increases with increasing cross-link density, whereas the colloid osmotic pressure (COP) decreases with increasing cross-link density. Interestingly, circular dichroism measurements indicate that the secondary structure of HSA is unaffected by polymerization. Altogether, these results show that glutaraldehyde can effectively cross-link HSA to produce high MW polymers, yielding a novel series of potential PEs that exhibit low COP and high viscosity.     

Relationship between in vitro growth promotion and biophysical and biochemical properties of the serum supplement

Summary The normal range of many chemical, physical, and biological (hormonal) properties of fetal bovine, calf, and newborn calf sera collected commercially are presented. We attempt to relate these properties to growth promotion in vitro. Whereas no definitive conclusions could be drawn from the data, high growth hormone levels, low endotoxin levels, and low levels of bilirubin seemed to have some association with good growth promotion if the levels of the other parameters were close to the mean. Large variations from the mean of any of the parameters seemed to affect the growth properties of the serum. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

Synthetic matrices reveal contributions of ECM biophysical and biochemical properties to epithelial morphogenesis

Epithelial cells cultured within collagen and laminin gels proliferate to form hollow and polarized spherical structures, recapitulating the formation of a rudimentary epithelial organ. However, the contributions of extracellular matrix (ECM) biochemical and biophysical properties to morphogenesis are poorly understood because of uncontrolled presentation of multiple adhesive ligands, limited control over mechanical properties, and lot-to-lot compositional variability in these natural ECMs. We engineered synthetic ECM-mimetic hydrogels with independent control over adhesive ligand density, mechanical properties, and proteolytic degradation to study the impact of ECM properties on epithelial morphogenesis. Normal cyst growth, polarization, and lumen formation were restricted to a narrow range of ECM elasticity, whereas abnormal morphogenesis was observed at lower and higher elastic moduli. Adhesive ligand density dramatically regulated apicobasal polarity and lumenogenesis independently of cell proliferation. Finally, a threshold level of ECM protease degradability was required for apicobasal polarity and lumen formation. This synthetic ECM technology provides new insights into how cells transduce ECM properties into complex morphogenetic behaviors.     

Characterization of the biochemical and biophysical properties of the phosphatidylserine receptor (PS-R) gene product

The PS-R gene product was originally described as a cell surface receptor that interacts with externalized phosphatidylserine (PS) on apoptotic cells, but more recent studies have shown that it plays a critical role in organ development and terminal differentiation of many cell types during embryogenesis. Despite these important developmental functions, the biochemical and molecular properties of PS-R are poorly understood. Here we have used several approaches to show that PS-R undergoes processive post-translational protein cross-linking to form covalent multimers within the nuclear compartment. Although PS-R has a potential Glu-Glu (QQ) duet that is often targeted by transglutaminase TG-2, the oligomerization of PS-R was not effected by QQ→AA mutation, or when PS-R gene product was expressed in TG-2 (-/-) fibroblasts. Pulse-chase experiments with ³⁵ S-methionine indicates that the PS-R undergoes an initial proteolytic cleavage, followed by progressive multimerization of the monomeric subunits over time. In summary, we report here that PS-R is modified by an unusual post-translational modification, and we speculate that homomultimer of PS-R might be playing an important function as a scaffolding protein in the nucleus.     

Comparative biochemical and biophysical studies on rat brain synaptosomes

A teichoic acid degrading enzyme (teichoicase) was purified to apparent homogeneity from a water-soluble cell extract of sporulating Bacillus subtilis cells. A rapid test for the detection of teichoicase activity was developed. The purified teichoicase has an app. Mr = 310 000. It consists of 4 identical subunits of Mr = 78 000 each.