Motor potential profile and a robust method for extracting it from time series of motor positions

Molecular motors are small, and, as a result, motor operation is dominated by high-viscous friction and large thermal fluctuations from the surrounding fluid environment. The small size has hindered, in many ways, the studies of physical mechanisms of molecular motors. For a macroscopic motor, it is possible to observe/record experimentally the internal operation details of the motor. This is not yet possible for molecular motors. The chemical reaction in a molecular motor has many occupancy states, each having a different effect on the motor motion. The overall effect of the chemical reaction on the motor motion can be characterized by the motor potential profile. The potential profile reveals how the motor force changes with position in a motor step, which may lead to insights into how the chemical reaction is coupled to force generation. In this article, we propose a mathematical formulation and a robust method for constructing motor potential profiles from time series of motor positions measured in single molecule experiments. Numerical examples based on simulated data are shown to demonstrate the method. Interestingly, it is the small size of molecular motors (negligible inertia) that makes it possible to recover the potential profile from time series of motor positions. For a macroscopic motor, the variation of driving force within a cycle is smoothed out by the large inertia.     

Prediction of the rotational diffusion behavior of biopolymers on the basis of their solution or crystal structure

Two low structure-resolution methods are proposed for prediction of rotational diffusion parameters. The indirect procedure is based on the structure of a molecule in solution or in crystal, and uses the structure parameters of radius of gyration, and low-resolution molecular surface and volume, determined from measured or theoretically calculated small-angle x-ray scattering intensities, to estimate a frictional equivalent ellipsoid of revolution. The direct method starts mainly from the crystallographic structure of a molecule and calculates the triaxial inertia equivalent ellipsoid, experimentally calibrated by translation diffusion data, to simulate the frictional behavior. The predicted harmonic mean of the rotational correlation times of compact globular macromolecules with molar masses of 14,000-65,000 g/mol agree with experimental results within the error limits. The prediction method is recommended for expert systems in structure research and for detection of internal protein flexibility or marker mobility by nmr and electron paramagnetic resonance experiments.     

Molecular imprinting of amino acid derivatives at low temperature (0 degrees C) using photolytic homolysis of azobisnitriles

Molecular imprints of L-phenylalanine anilide (print molecule) were prepared using a number of free radical initiation systems. Polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at temperatures ranging from 0 to 60 degrees C and evaluated in the high-performance liquid chromatographic mode for enantioselectivity. The results show that preparation of molecular imprints at 0 degrees C using photolytic homolysis of azobisnitriles significantly increases enantioselectivity and allows separations of the enantiomers of the print molecule to be performed at room temperature. This compares to previous reports of molecular imprints where separations needed to be performed at elevated temperatures (80-90 degrees C). The present method is also easier to perform and less time-consuming than those previously described.     

Amino acid makeup, structural characteristics and substrate specificity of rabbit plasma kininogen]

Highly purified kininogen preparation with the activity of 16-18 int. units per mg was isolated from rabbit blood serum. Its molecular weight was estimated to be 54 000 by gel filtration through Sephadex G-200. Leucine was identified as N-terminal amino acid by the dansylation method. Rabbit kininogen consists of 394 amino acid residues (except tryptophane). Amino acid composition of kininogen is characterized by a high content of dicarbonic amino acids, proline and by a low content of methionine. Kininogen molecule does not contain SH-groups. 13.1-13.5 SH-groups were found in kininogen after the reduction of S-S bonds with beta-mercaptoethanol in the presence of 8 M urea, thus indicating the presence of 6-7 S-S bonds in kininogen molecule. Kininogen group does not occupy C-terminal position in the molecule, because the treatment of the protein with carboxypeptidase B does not change the content of bradykinine in it. Purified kininogen preparation is a substrate for kallikrein from rabbit blood plasma, human saliva and trypsin. Unlike trypsin, kallikreines from human blood plasma and saliva release kinines from kininogen with reduced S-S bonds. Under spontaneous reoxidation of reduced S-S bonds up to 90%, substate properties of kininogen for tripsin recover only by 50%. Rabbit kininogen is similar to beef kininogen II in its molecular weight, amino acid composition and the number of S-S bonds.     

The structure of bactoprenol, a lipid formed by lactobacilli from mevalonic acid

1. The name `bactoprenol' has been given to the most abundant lipid formed by three species of lactobacilli from mevalonic acid. 2. A method for the preparation of pure bactoprenol is described. 3. The thin-layer chromatographic properties of bactoprenol and of its acetylated and hydrogenated derivatives resembled those of dolichol. 4. Analysis by mass spectrometry and by nuclear magnetic resonance showed that the molecule is formed by condensation of 10 unsaturated isoprene units and 1 saturated isoprene unit. 5. Its molecular weight is 768 and it has 10 double bonds/molecule. 6. Infrared spectroscopy and the uptake of acetyl groups indicated that the molecule contains a hydroxyl group. 7. It is concluded that bactoprenol is a C₅₅ isoprenoid alcohol.     

Jump rates for anisotropic particles

A theoretical analysis for site-to-site jump rates for anisotropic molecules is presented. The molecular shape is regarded as a mechanical anisotropy in the form of finite moments of inertia, as well as anisotropy with respect to the interaction potential. The mutual coupling between rotational and translational motion necessarily produces a competitive effect between the equilibrium alignment in the local field and the precession of the figure axis, leading to an increase of the effective activation energy. As a numerical example the jump rate for a water molecule in a gramicidin-like channel has been calculated, and a temperature-independent reduction of some 15% for the rate as compared to the point-like molecule has been found.     

Mechanism of local anesthesia: long-range orientation effects

The orientation interaction of a molecule of a local anesthetic with a biomembrane and cell-like liquid was studied, based on the model of adsorption of the anesthetic from a cell-like solution on the surface of a biomembrane for compounds of the trimecaine series. A statistically significant correlation equation was obtained, which relates the minimum blocking concentration to the projection of the dipole moment of the anesthetic on the plane X(1)0X2 of the principal axes of inertia. A model is prosed according to which the "anesthetic-biomembrane" interaction is most effective the molecule of the anesthetic rotates around the axis of the maximum moment of inertia.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.     

Proteolytic degradation of ferredoxin-NADP reductase during purification from spinach

Ferredoxin-NADP reductase (FNR) was rapidly isolated from spinach leaves with special care to suppress proteolytic degradation. The molecular mass of this FNR preparation was estimated to be 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Limited proteolysis of 35-kDa FNR to 33-kDa FNR was effectively suppressed by high pH (at pH 9.3), concentrated salts, and low temperature. On the basis of these observations, a new isolation procedure was designed to obtain 35-kDa FNR in a preparative scale. The resulting final preparation still contained two FNR components. One appeared to correspond to the longest polypeptide so far reported for spinach FNR (Karplus et al., 1984, Biochemistry 23, 6576-6583) while the other lacked a gamma-pyroglutamyl residue from its amino terminus. Conventional preparation procedure without suppression of proteolytic action yielded an FNR preparation with a molecular mass of 33 kDa. This FNR preparation consisted of three components. They lacked 11 to 17 amino-terminal residues, while their carboxyl-terminal structure was retained intact. These results showed that proteolytic degradation of the spinach FNR molecule during purification took place exclusively at its amino-terminal moiety and further suggested that 35-kDa FNR with Karplus' structure should be the mature FNR molecule functional in the chloroplast thylakoids.     

Selective removal of heparan sulfate chains from proteoheparan sulfate with a commercial preparation of heparitinase

Procedures employing the commercial preparation of heparitinase were developed for isolating a protein-enriched core molecule from proteoheparan sulfate by selective removal of the heparan sulfate chains. Treatment of proteoheparan sulfate with the enzyme preparation caused seriously extensive degradation owing to the presence of proteolytic activity in the enzyme preparation. This effect could be avoided by using a series of protease inhibitors which prevented proteolytic degradation with less significant effect on the heparitinase activity. Application of the procedures to a purified preparation from the Engelbreth-Holm-Swarm tumor yielded a single protein-enriched core fraction with a molecular weight of approximately 450,000, as ascertained by sodium dodceyl sulfate-gel electrophoresis.     

Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.     

Molecular size heterogeneity of human leukocyte interferon

Molecular sieving of human leukocyte interferon revealed an apparent molecular weight of 26,000. However, after denaturation by guanidine hydrochloride in the presence of a reducing agent and reactivation by extensive dialysis, a molecular weight of only 21,000 was observed. The reactivated human leukocyte interferon (mol wt 21,000) gave a single peak of activity when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that a single molecular weight species was generated by the denaturation and reactivation procedure. A partial unfolding of the molecule was evident when the interferon preparation was heated to 50 degrees C in the absence or presence of an unfolding agent and then sieved on Sephadex G-100 Superfine. These results suggest that the interferon molecule undergoes a proteolytic cleavage probably by a protease present in extracellular fluid. Thus, a peptide fragment dissociates from the parent molecule when human leukocyte interferon is denatured in the presence of a reducing agent, resulting in a drop of 5,000 in molecular weight; interestingly, the resultant 21,000 molecular weight form still retains its antiviral activity.     

Absorption of x-radiation by single crystals of proteins containing labile metal components: the determination of the number of iron atoms within the central core of ferritin

The number of metal atoms contained within a displaceable inorganic component of a metalloprotein was determined by considering X-ray absorption by single crystal samples of holo- and apo-proteins. Since this method is non-destructive, it can be used to determine the number of metal atoms associated with the molecules forming the crystal actually used for X-ray diffraction data collection and subsequent structure solution. The method has been applied to the iron storage protein ferritin, isolated from horse spleen, to give a reliable estimate of the average iron content of the ferritin molecules within the crystal. This value, of around 2000 iron atoms per molecule is consistent with that found for a typical ferritin preparation in solution and suggests non-selectivity of the crystallisation process for ferritin in terms of molecular iron content.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

Carboanhydrase from bean leaves

A highly purified preparation of carboanhydrase from the leaves of the bean Vicia faba var. major (Harz) F. Janthina was obtained. The enzyme was homogeneous during analytical disc-electrophoresis in polyacrylamide gel and analytical ultracentrifugation. The molecular weight of the enzyme is 270,000. The enzyme molecule contains six Zn atoms, has a quaternary structure and is made up of six subunits with molecular weights of 45,000. Some properties of the enzymes from bean leaves and pea leaves were compared. The enzymes differ in molecular weights and behaviour during DEAE-cellulose chromatography and gel filtration through Sephadex G-200.     

Study of a protein complex from the brain which reduces actin viscosity. I. Composition and various properties of the complex

The method of isolation from bovine brain of a preparation containing 90 kDa- and 42 kDa-proteins is described. This preparation shortens actin filaments and therefore decreases viscosity of F-actin. The 42 kDa-component was identified as actin by one-dimensional peptide mapping. Quantitative densitometry has demonstrated that 90 kDa-protein and actin are present in the preparation in equimolar ratio. Fractionation of the preparation by gel-filtration, analytical centrifugation or electrophoresis under non-denaturing conditions showed that 90 kDa-protein and actin are in a light complex. This complex consists of one actin molecule and one molecule of 90 kDa-protein and has a sedimentation coefficient of 3.5S. Both beta- and gamma-isoelectric forms of actin are present in the complex.     

Purification and molecular properties of vitellin from the silkworm,Bombyx mori

Vitellin was purified from the eggs of the silkworm, Bombyx mori by a simple method which included a specific precipitation at pH 6 under low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy and ultracentrifugation.Vitellin was defined as glycolipoprotein with a sedimentation coefficient (S_{20, W}) of 13.5S and a molecular weight of 440,000. The molecule was almost spherical in shape with a diameter of 13 nm. The molecule contained 3% mannose and 7.5% total lipids which comprised triacylglycerol, diacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The amino acid composition displayed a high content of glutamic and aspartic acids and a low content of methionine. The molecule was composed of two non-identical subunits with molecular weights of 180,000 and 42,000, and the native molecule was assumed to be a tetramer composed of two molecules of each of these subunits. Separation of the two subunits was achieved, and mannose was covalently associated only with the heavier subunit.The rabbit anti-egg vitellin antibody cross-reacted with the haemolymph vitellogenin but not with other haemolymph proteins, nor with the vitellogenin from Locusta migratoria. The antibody also reacted with the haemolymph vitellogenin of the silkworm, Philosamia cynthia.     

Characterization and properties of Pholas luciferase as a metalloglycoprotein.

The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.     

Isolation and characterization of the human adenocarcinoma-associated glycoprotein gp40

The human adenocarcinoma-associated antigen gp40 is a cell surface glycoprotein recognized by murine monoclonal antibody KS1/4. A KS1/4-Sepharose affinity matrix was utilized to purify gp40 from detergent lysates of either tissue culture cells or nude mouse xenograft tumors of the human lung adenocarcinoma cell line P3-UCLA. This single immunoaffinity chromatography step yielded an antigen preparation of approximately 95% purity which was further characterized by immunochemical and enzymatic techniques. The gp40 molecule was shown to have both complex and high-mannose oligosaccharides comprising some 16% of the apparent molecular weight. The antigen preparation was suitable for gas-phase N-terminal amino acid sequencing and the first 16 residues of the N-terminus were determined. Despite considerable molecular heterogeneity, gp40 shows a single N-terminal sequence.     

Preparation of red-cell-membrane cytoskeletal constituents and characterisation of protein 4.1

A new and rapid method is described for the preparation of protein 4.1, the protein which modulates the interaction between spectrin and actin in the membrane cytoskeleton of the red cell. The method is based on the dissociation of purified membrane cytoskeletons in concentrated Tris at neutral pH, followed by gel filtration in the same medium. This procedure also yields spectrin and actin, as well as the fourth cytoskeletal constituent, protein 4.9, in relatively pure form, and ankyrin. Protein 4.1 is monomeric under our conditions of solvent and protein concentration, with a relative molecular mass, as determined from sedimentation equilibrium, of about 78 000; its sedimentation coefficient and Stokes' radius are those of a globular, though somewhat asymmetric or flexible molecule. It forms a strong complex with F-actin and spectrin. Protein 4.9 is also recovered in active form, and will bind strongly to F-actin.