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31.
Spooner PJ Sharples JM Verhoeven MA Lugtenburg J Glaubitz C Watts A 《Biochemistry》2002,41(24):7549-7555
Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ). 相似文献
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A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid. 相似文献
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Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems. 相似文献
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Vandenberg R Miller J Ugarte M Kardolus J Villand J Nienhuis J Spooner D 《American journal of botany》1998,85(1):92-109
The major cultivated potato, Solanum tuberosum, and six other related cultivated species, are hypothesized to have arisen from a group of weedy relatives indigenous to the central Andes of central Peru, Bolivia, and northern Argentina. A major problem hindering investigations of the origins of the cultivated species has been a continuing debate over the species boundaries of their putative progenitors. This study investigated the morphological phenetic species boundaries of these putative progenitors and five cultivated taxa, here collectively referred to as the Solanum brevicaule complex. Two hundred fifteen accessions of 30 taxa in the S. brevicaule complex and 42 accessions of six taxa outside of the complex were assessed for 53 morphological traits in replicate plots in a common garden, resulting in a total of over 81;t3000 data points. Phenetic analyses of these data are unable to support 30 taxa, suggesting instead a single variable complex at best only weakly divided into three widely intergrading sets of populations: (1) Peruvian and geographically adjacent Bolivian accessions (including wild species and all the cultigens), (2) Bolivian and Argentinian accessions and S. verrucosum from Mexico (including only wild species), and (3) the Bolivian and Argentinian wild species S. oplocense. These and other data suggest that Hawkes's 1990 treatment (The Potato: Evolution, Biodiversity, and Genetic Resources, Smithsonian Institute Press, Washington, DC.) of 232 morphological species is an overestimate for sect. Petota. 相似文献
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Aim There has been considerable debate about pre‐settlement stand structures in temperate woodlands in south‐eastern Australia. Traditional histories assumed massive tree losses across the region, whereas a number of recent histories propose that woodlands were originally open and trees regenerated densely after settlement. To reconcile these conflicting models, we gathered quantitative data on pre‐settlement stand structures in Eucalyptus–Callitris woodlands in central New South Wales Australia, including: (1) tree density, composition, basal area and canopy cover at the time of European settlement; and (2) post‐settlement changes in these attributes. Location Woodlands dominated by Eucalyptus species and Callitris glaucophylla, which originally occupied approximately 100,000 km2 in central New South Wales, Australia. Methods We recorded all evidence of pre‐settlement trees, including stumps, stags and veteran trees, from 39 relatively undisturbed 1‐ha stands within 16 State Forests evenly distributed across the region. Current trees were recorded in a nested 900 m2 quadrat at each site. Allometric relationships were used to estimate girth over bark at breast height, tree basal area, and crown diameter from the girth of cut stumps. A post‐settlement disturbance index was developed to assess correlations between post‐settlement disturbance and attributes of pre‐settlement stands. Results The densities of all large trees (> 60 cm girth over bark at breast height) were significantly greater in current stands than at the time of European settlement (198 vs. 39 trees ha?1). Pre‐settlement and current stands did not differ in basal area. However, the proportional representation of Eucalyptus and Callitris changed completely. At the time of settlement, stands were dominated by Eucalyptus (78% of basal area), whereas current stands are dominated by Callitris (74%). On average, Eucalyptus afforded 83% of crown cover at the time of settlement. Moreover, the estimated density, basal area and crown cover of Eucalyptus at the time of settlement were significantly negatively correlated with post‐settlement disturbance, which suggests that these results underestimate pre‐settlement Eucalyptus representation in the most disturbed stands. Main conclusions These results incorporate elements of traditional and recent vegetation histories. Since European settlement, State Forests have been transformed from Eucalyptus to Callitris dominance as a result of the widespread clearance of pre‐settlement Eucalyptus and dense post‐settlement recruitment of Callitris. Tree densities did increase greatly after European settlement, but most stands were much denser at the time of settlement than recent histories suggest. The original degree of dominance by Eucalyptus was unexpected, and has been consistently underestimated in the past. This study has greatly refined our understanding of post‐settlement changes in woodland stand structures, and will strengthen the foundation for management policies that incorporate historical benchmarks of landscape vegetation changes. 相似文献
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