Exhaustive enumeration of protein conformations using experimental restraints.

We present an efficient new algorithm that enumerates all possible conformations of a protein that satisfy a given set of distance restraints. Rapid growth of all possible self-avoiding conformations on the diamond lattice provides construction of alpha-carbon representations of a protein fold. We investigated the dependence of the number of conformations on pairwise distance restraints for the proteins crambin, pancreatic trypsin inhibitor, and ubiquitin. Knowledge of between one and two contacts per monomer is shown to be sufficient to restrict the number of candidate structures to approximately 1,000 conformations. Pairwise RMS deviations of atomic position comparisons between pairs of these 1,000 structures revealed that these conformations can be grouped into about 25 families of structures. These results suggest a new approach to assessing alternative protein folds given a very limited number of distance restraints. Such restraints are available from several experimental techniques such as NMR, NOESY, energy transfer fluorescence spectroscopy, and crosslinking experiments. This work focuses on exhaustive enumeration of protein structures with emphasis on the possible use of NOESY-determined distance restraints.     

Transbilayer distribution of phospholipids in photoreceptor membrane studied with trinitrobenzenesulfonate alone and in combination with phospholipase D

In a further study of the transbilayer distribution of phospholipids in rod disk membranes, the amino group reagent, trinitrobenzenesulfonate, and the phospholipid-hydrolyzing enzyme, phospholipase D, have been used alone and in combination.Under carefully defined conditions (1 mM trinitrobenzenesulfonate, pH 7.4, 20°C, darkness), trinitrobenzenesulfonate yields limited final levels of modification of phosphatidylethanolamine and phosphatidylserine, suggesting only minor reagent penetration and membrane disturbance under these conditions.Treatment of stacked disks with trinitrobenzenesulfonate under these conditions leads to a biphasic modification of the a aminophospholipids. Relatively fast (less than 1 h) modification of 50% phosphatidylethanolamine and 40% phosphatidylserine occurs, slowly rising (approx. 3 h) to 60 and 50%, respectively.Extensive treatment of stacked disks with phospholipase D leads to the hydrolysis of 55% phosphatidylcholine and 50% phosphatidylethanolamine, while phosphatidylserine is hardly attacked by this enzyme.Treatment of stacked disks with trinitrobenzenesulfonate after prior treatment with phospholipase D leads to no further modification than that maximally obtained with either reagent alone: about one-half of the three major phospholipid classes is accessible. Although both reagents differ greatly in molecular size, mode of action and other properties, they apparently see the same pool of phosphatidylethanolamine, their joint substrate. Considering that we start with the original right-side-out configuration, that all phospholipids can in principle be modified (no shielding) and that the membrane remains essentially intact, we conclude that the accessible lipid pool represents the outer face of the disk membranes.These results confirm our earlier conclusions from treatment with three phospholipases that the three major phospholipids are nearly symmetrically distributed over the two faces of the disk membrane.The divergence with the conclusions of other investigators is most likely explained by their use of disk membranes (disk vesicles) in which the original phospholipid distribution had not been maintained and/or of conditions under which trinitrobenzenesulfonate markedly penetrates the membrane.     

Effects of abscisic acid,gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.Abbreviations ABA abscisic acid - FC fusicoccin - GA₃ gibberellic acid - PD electric potential difference (between the vacuole and the external medium) - CH cycloheximide - DNP dinitrophenol - FCCP (p-trifluormethoxy)-carbonylcyanide-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide     

Fine structure of the gap junction in the tunicate heart

Summary The plasma membranes of the tunicate heart exhibit an abundance of macular gap junctions distributed widely over the membrane surface. A study of these junctions by the freeze-etch technique was undertaken in an effort to elucidate the fine structure of this important membrane modification in a primitive heart. In cross or near-cross fractured junctions the junctional particles in contiguous membranes appear to be paired in register and to meet in the midline. In favorable face views, the junctional particles are seen to be disposed in hexagonal array. The individual particles display a distinct rosette-like substructure consistent with a six-membered ring of globular protein molecules clustered around a central channel. Similar junctional-type particles can be found in nonjunctional areas of membrane suggesting that the transport mechanism which they may represent is not restricted to the gap junction.Career Investigator of the American Heart AssociationWe wish to thank Dr. J.B. Jillett for use of the facilities of the Portobello Marine Biological Station; Mr. W.S. Bertaud, Physics and Engineering Laboratory, D.S.I.R., Lower Hutt, who kindly supervised the preparation of some of the freeze-etch replicas; Dr. R.H. Millar of the Dunstaffnage Marine Research Laboratory, Oban, Argyll, Scotland, who identified the tunicate used in the present (and previous) study; Prof. W.D. Trotter who made facilities in the Department of Anatomy, University of Otago Medical School, Dunedin, available to one of us (V.L.); and Mrs. S.M. O'Kane for excellent technical assistance. Generous support from the American Heart Association (to V.L.) and from the Medical Research Council of New Zealand (to D.G.R.) is gratefully acknowledged     

Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

Articular and diaphyseal remodeling of the proximal femur with changes in body mass in adults.

Proximal femoral dimensions were measured from radiographs of 80 living subjects whose current body weight and body weight at initial skeletal maturity (18 years) could be ascertained. Results generally support the hypothesis that articular size does not change in response to changes in mechanical loading (body weight) in adults, while diaphyseal cross-sectional size does. This can be explained by considering the different bone remodeling constraints characteristic of largely trabecular bone regions (articulations) and largely compact cortical bone regions (diaphyses). The femoral neck shows a pattern apparently intermediate between the two, consistent with its structure. When the additional statistical "noise" created by an essentially static femoral head size is accounted for, the present study supports other studies that have demonstrated rather marked positive allometry in femoral articular and shaft cross-sectional dimensions to body mass among adult humans. Body weight prediction equations developed from these data give reasonable results for modern U.S. samples, with average percent prediction errors of about 10%-16% for individual weights and about 2% for sample mean weights using the shaft dimension equations. When predicting body weight from femoral head size in earlier human samples, a downward correction factor of about 10% is suggested to account for the increased adiposity of very recent U.S. adults.     

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [¹⁴C]methylamine. The pH of unfractionated blood cells is 7.39±0.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.     

The fine structure of the terminal segment of the bovine seminiferous tubule

Summary The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.Supported by a grant from the Deutsche Forschungsgemeinschaft     

The relation between nitrogen percent and dry weight of inoculated legumes

Summary Results of numerous tests of tropical and temperate legume hosts and Rhizobium strains accumulated between 1952 and 1966 were examined for a relation between N percent (y) and dry weight per plant (x). The data fitted the equation y=A–be^–cx. The most effective Rhizobium strains can be selected on the basis of dry matter yields of whole plants or plant tops only, without the need for N analyses, using statistical analyses. A previously proposed method which employs the linear relation between N-yield and dry matter yield was shown to apply only when data for strains which are not fully effective are excluded.It is postulated that symbiotically fixed N forms a different compound from that resulting from applied mineral N and that this compound cannot be remobilised before flowering.deceased