Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.

␣-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC 50 ‫؍‬ 0.1 mM. The inhibition type for RPE65 was found to be uncompetitive with K i ‫؍‬ 53 M. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-␣-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.In several previous publications, we have shown that ␣-phenyl-N-tert-butylnitrone (PBN) 4 (see Fig. 1), a commonly used free radical spin trap, provides remarkable protection of photoreceptor and RPE cells from light-induced damage (1-5). Because of its anti-oxidant properties and based on several reports demonstrating beneficial pharmacological effects, including reduction in mortality associated with endotoxin shock (6 -8), neuroprotection in ischemia-reperfusion and aging models (9 -10), and prevention of streptozotocin-induced diabetes in mice (11), we speculated that PBN could be a useful therapeutic intervention against retinal degenerative diseases, such as age-related macular degeneration. Because the retina has a high oxygen demand, is chronically exposed to light, and contains several photosensitizers, oxidative stress is presently considered to be a cause of disease progression in age-related macular degeneration (12). Thus, we speculated that PBN, which is already known to be effective against agerelated and accumulative oxidative stress (13, 14), might also be effective against age-related macular degeneration. The mechanism(s) underlying the PBN-mediated protection of photoreceptor cells are not well understood. Given the role of oxidative stress in retinal light damage (15-18) and the free radical scavenging properties of PBN, it seemed logical to propose that PBN exerts antioxidant function i...

The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADPVi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-P,.Muscle contraction and many other manifestations of biological motility are based on the cyclic interaction of myosin heads with actin, which is accompanied by ATP hydrolysis. During steady-state ATP hydrolysis the myosin head is subjected to conformational changes that can be detected by changcs in ultraviolet absorption Enzyme. Chymotrypsin (EC 3.4.21 .I).is used as a stable analogue of the S1-ATP complex.Complexes of S1 with ADP and orthovanadate (Vi) [S, 61 and S1 cross-linked by p-phenylene-N,N-dimaleimide (pPDM) in the presence of ADP [7] are often used as stable analogues of the S1-ADP-Pi complex. The use of stable analogues allows the structure and properties of S1 in the intermediates of the ATPase reaction to be studied. There are numerous data suggesting that the stable analogues of the S1-ATP and S1-ADP-Pi intermediates are similar in structure, but differ from nucleotide-free S1 and S1-ADP [8-201, It should be noted that all these data are in fact indirect since they are based on either studies of the actinbinding properties of S1 [8 -151 or studies of local conformational changes in S1 [16-201. In order to obtain direct proof of the assumption of two main structural states of S1, one must know the effects of nucleoti...

Tropomyosin (Tm) is a two-stranded ␣-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle ␣-Tm either with an Ala residue, which should stabilize the Tm ␣-helix, or with an Arg residue, which is expected to stabilize both ␣-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60 -70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a singlespanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.Proteins of the Bcl-2 family are key regulators of apoptosis that function either as promoters or inhibitors and that display homology in one to four short sequences termed Bcl-2 homology (BH) 3 motifs (1-3). Anti-apoptotic subfamily proteins such as Bcl-2 and Bcl-x L contain four BH motifs (BH1-4). Pro-apoptotic proteins are grouped into either multiple or single BH motif subfamilies. The former subfamily includes the proteins Bax and Bak that display homology in BH1-3 motifs. The latter includes proteins such as Bid, Bim, and Bad that are similar only in the limited BH3 motif. Despite the limited sequence homology, the three-dimensional structures determined for seven Bcl-2 family proteins including members from all three subfamilies are very similar, consisting of a hydrophobic core of one to three helices wrapped by five to eight amphipathic helices and their connecting loops. This structure is strikingly similar to the structure of the pore-forming domains of diphtheria toxin and Escherichia coli colicins. In addition there is an obvious hydrophobic groove on the surface of all of the anti-apoptotic Bcl-2 family proteins (4 -6). From decades of extensive studies, two properties are well known to be shared by most, if not all, Bcl-2 family proteins: homo-or hetero-binding and pore formation in membranes. More experiments have been done on the homo-or heterobinding of Bcl-2 family p...