The combinatorial distance geometry method for the calculation of molecular conformation. I. A new approach to an old problem

A new approach to the long-standing local minimum problem of molecular energy minimization is proposed. The approach relies upon a field of computer mathematics known as combinatorial optimization, together with methods of conformational analysis derived from distance geometry. The advantages over the usual numerical techniques of optimization are, first, that the algorithms derived are globally convergent, and second, that the mathematical problems involved are well-posed and suitable for study within the modern theory of computational complexity. In this paper we introduce the approach, and describe a computer program based on it.     

A computer program for processing data from amino acid analysis and for the calculation of molecular weights from those data.

A computer program is described for the calculation of the complete amino acid composition of a protein from the analytical data. The program also derives a molecular weight on the basis of the amino acid composition. The use of the program for the determination of the molecular weights of the liver carboxylesterases of chicken, horse, ox, and sheep is described.     

The structure and conformation of HC-toxin

Difference nuclear magnetic resonance studies and amino acid oxidase experiments establish the sequence and configuration of amino acids in HC-toxin as cyclo(L-Aoe-D-Pro-L-Ala-D-Ala). HC-toxin adopts the bis-γ-turn conformation in solution previously found for the cytostatic cyclic tetrapeptide chlamydocin.     

Thyroid hormone structure: molecular conformation of 3'-isopropyl-3,5-diiodo-L-thyronine, the most potent known thyromimetic agent

The three-dimensional structure of the potent thyromimetic agent 3′-isopropyl- 3,5-diiodo-L-thyronine (iPr-T₂) has been established by x-ray diffraction of single crystals of iPr-T₂ hydrochloride. The molecular conformation is such that the β-ring 3′-isopropyl group is oriented in space $\text{proximal}$ to the 3,5-diiodotyrosine α-ring, similar to the conformation adopted in the crystal structure of 3,5,3′-triiodo-L-thyronine.     

Studies on the conformation of secretin: The position of the helical stretch

An analog of the C-terminal tricosapeptide of secretin, with aspartic acid replacing glutamic acid in position 9 and lysine substituted for arginine in position 21, was prepared. The synthesis was carried out in solution by stepwise chain lengthening with the application of the in situ technique. The ord-cd spectra of this new analog closely resemble the spectra of the tricosapeptide with the unaltered secretin sequence and of the analog in which only arginine-21 was replaced by lysine and of secretin itself. The incorporation of aspartic acid instead of glutamic acid-9 resulted in an N-terminal sequence that has a consïderably reduced probability of assuming a helical conformation. The observation that the helix content remained unchanged adds support to a model of secretin in which the helical stretch is near the C-terminus. The role of an acidic residue in position 9 is also discussed.     

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenylphosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), γ-glutamyltransferase (EC 2.3.2.2), $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase ($\text{acyl-CoA:1-acylglycero-3-phosphocholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution.These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.     

X-ray crystal and molecular structures of the Ni(II) and Co(II) complexes of 2'-deoxyguanosine-5'-monophosphate.

The structures of [Ni(5′-dGMP)(H₂O)₅] and [Co(5′-dGMP)(H₂O)₅] have been solved by single-crystal x-ray diffraction techniques. Their common geometry consists of a metal ion octahedrally coordinated to the N₇ atom of guanine and five water ligands. The phosphate group of the nucleotide is hydrogenbonded to two of the coordinated water molecules.     

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.     

The alpha-glucosidases of Dictyostelium discoideum. II. Developmental regulation and cellular localization

Four isozymes of α-glucosidase in Dictyostelium discoideum have been identified and some of their enzymatic and physical properties characterized (R. H. Borts and R. L. Dimond, 1981, Develop. Biol.87, 176–184). In this report the cellular localization and developmental regulation of three of these isozymes are determined. α-Glucosidase-1 is the major isozyme of vegetative amoebae. It is lysosomally localized and secreted from the cell under certain conditions. It has an acidic pH optimum and carries the common antigenic determinant found on all lysosomal enzymes in this organism. The specific activity of this isozyme begins to decrease within a few hours after the initiation of development and is no longer detectable in the mature fruiting body. α-Glucosidase-2 has a neutral pH optimum and is neither lysosomal nor secreted. Rather it is membrane bound and is possibly located on the cisternal side of microsomal vesicles. This isozyme does not possess the common antigenic determinant. α-Glucosidase-2 comprises 20–40% of the total α-glucosidase activity of the vegetative cell. Its specific activity increases threefold during development. This isozyme appears to be developmentally controlled since it fails to accumulate in aggregation deficient mutants. Its accumulation is also dependent upon continued protein synthesis. α-Glucosidase-4, like α-glucosidase-1, has an acidic pH optimum. It does not appear to be lysosomally localized nor membrane bound. Approximately 30% of the activity is precipitable by antibody against the common antigenic determinant indicating that it is less highly modified or fewer molecules are modified. The isozyme is undetectable during vegetative growth and does not begin to accumulate until late aggregation. Activity peaks in mature fruiting bodies where it is the predominant acidic α-glucosidase activity. Accumulation of α-glucosidase-4 is blocked in morphologically deficient mutants and by inhibitors of protein synthesis.     

Chemical synthesis of versatile adaptors for molecular cloning.

In this communication we report the chemical synthesis of two types of oligodeoxynucleotides to be used as adaptors in molecular cloning. The first type is used to create specific cohesive end sequences at the termini of a blunt-end DNA molecule without the use of restriction enzymes. The second type of adaptor is used to convert one kind of restriction-enzyme-generated specific cohesive end to another. This includes both the conversion of one type 5′-protruding end to another 5′-protruding end, and of a 3′- to a 5′-protruding end and vice versa.     

Development of the rat salivary glands. II. The identification of a trypsinlike protease in the submaxillary gland

Trypsinlike protease activity at pH 9.2 was measured in tissue extracts of adult rat salivary glands by using a fluorometric assay in which β-naphthylamine is released by the hydrolysis of benzylarginine β-naphthylamide. The submaxillary gland contains high levels of this activity, and the parotid and sublingual glands have a maximum of 2000-fold and 200-fold less. After polyacrylamide disc gel electrophoresis at pH 8.3, the protease activity of submaxillary extracts is associated with a major protein band. Neither this protein band nor its protease activity is detectable in extracts of parotid or sublingual glands. Homogenates of newborn submaxillary gland do not have this protease activity at detectable levels, suggesting that its major accumulation is postnatal.     

An improved method for the separation of peptides and alpha-amino acids on copper-Sephadex.

An improved method for the separation of peptides from large amounts of α-amino acids on copper-Sephadex is described. The separation is essentially dependent upon the copper content of Sephadex, the pH of the system, and the concentration of the sample, and it is due to the different stabilities of copper complexes of Sephadex, peptides, and α-amino acids. Sephadex behaves as a solid ligand. Compounds that form weaker complexes with copper than Sephadex apparently move with the solvent front. α-Amino acids that form copper complexes having a stability comparable to that of copper-Sephadex complexes are retained on the column. Peptides form strong complexes, stripping copper from the column. They are only slightly retained. The method is most practical when copper is removed from the eluates with chelating ion-exchanger Dowex A-1. Examples are given for the chromatography of single compounds, model mixtures, and extracts from cheese and yeast.     

Immunocapillarymigration--a new method for immunochemical quantitation.

A new simple and rapid method for immunochemical quantitation called immunocapillarymigration is described. It is based upon the attachment of antibodies to a porous insoluble support and the subsequent capillarymigration of the antigen-containing solution in the porous support. The migration of the antigen solute is specifically delayed in comparison to the migration of the solvent and other solutes in the process and the relative delay decreases with increasing antigen concentration. When applied to the quantitation of transferrin in human plasma, immunocapillarymigration gave results which agreed with those obtained by single radial immunodiffusion.     

A new method for the determination of N-sulfate in heparin and its analogs.

A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.     

First observation of a beta-turn conformation fused with the oxy-analogue of an alpha-turn: the molecular structure of a model peptide of the C-terminal part of gramicidin A

The molecular structure of N-tert-butyloxycarbonyl-D-leucyl-L-phenylalanyl ethanolamide (t-Boc-D-Leu-L-Phe-EA), a protected analogue of the C-terminal dipeptide of the membrane-active linear antibiotic gramicidin A, has been determined by X-ray diffraction. One of the two independent molecules in the asymmetric unit is characterized by a chain reversal stabilized by an intramolecular, three-centre, double hydrogen bonding. It represents the first experimental evidence for a beta-turn conformation fused with the oxy-analogue of an alpha-turn.     

Calcium-induced changes in thyroglobulin conformation

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.     

Polymer conformation: 2. Rapid methods for locating contact-free molecular structures with particular reference to sidegroup interactions,linkage maps and crystal packing of rigid helical chains

Paper 1 in this series¹ outlined a method for identifying sets of backbone conformations of a polymer which are of a given helical type. This paper is concerned with fast methods for the calculation and mapping of contact-free structures in cases where the structure consists of two rigid molecular groups and where the position of each group varies by rotation about its own distinct axis. In particular, it is shown how such methods can be used to locate the neighbourhoods of crystal packing minima for a helical polymer chain, which could be generated by the method of Paper 1 and which is to be packed rigidly into a lattice. The method does not possess the flexibility of a full refinement procedure, but its advantages, apart from its conjectured speed, include an exactly known level of exhaustion of the examined variables and immunity to false packing minima and choice of initial starting position. Together, the methods may provide a means for checking the capability of refinement procedures to locate packing minima.     

On the multiplicity of platelet prostaglandin receptors: II. The use of N-0164 for distinguishing the loct of action for PGI2, PGD2, PGE2 and hydantoin analogs

The ω-chain variant analogs of prostacyclin (PGI₂) and PGD₂ in which the n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1)(1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI₂, but converts PGD₂ to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD₂ by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD₂, cyclohexyl-PGD₂, and BW-245C; with essentially no effect on PGI₂, cyclohexyl-PGI₂ and PGE₂ at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre (2,3), but supports the view that the anti-aggregatory effect of high doses of PGE₂ ($\text{EC}{}_{50}\text{=50μ}\text{M}$) is mediated by the PGI₂ receptor (4). The hydantoin acts at the platelet PGD₂ receptor.     

The double-helical molecular structure of crystalline b-amylose

The crystal structure of the B-polymorph of amylose appears to be based on double-stranded helices. The individual strands are in a right-handed six-fold helical conformation repeating in 20.8 Å and are wound parallel around each other. The steric disposition of O-6 is gt. The double helices pack in a hexagonal unit-cell (a  b  18.50 Å, c (fiber repeat)  10.40 Å, γ  120°), with two helices (12 d-glucose residues) per cell. The helices are packed antiparallel and leave an open channel within a hexagonal array that is filled with water molecules. The reliability of the structure analysis is indicated by R  0.22. The structure of B-amylose is consistent with the diffraction diagrams of B-starches and accounts for the physical properties of such starches.     

A rapid method for separating small vesicles from suspension.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.