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R A Mendelson

Publications and source records attributed to R A Mendelson.

At least 19 recordsLinked to original sources
PubMed1999

Solution structures of dimeric kinesin and ncd motors.

The dimeric structure of the members of the kinesin family of motor proteins determines the individual characteristics of their microtubule-based motility. Crystal structures for ncd and kinesin dimers, which move in opposite directions on microtubules, show possible states of these dimers with ADP bound but give no information about these dimers in solution. Here, low-angle X-ray and neutron scattering were used to investigate their solution structures. Scattering profiles of Drosophila ncd 281-700 (NCD281) and human kinesin 1-420 (hKIN420) were compared with models made from the crystallographically determined structures of NCD281 and rat kinesin 1-379 (rKIN379). From the low-angle region it was found that the radius of gyration (Rg) of NCD281 is 3.60 +/- 0.075 nm, which is in agreement with the crystallography-based model. Scattering by longer ncd constructs (NCD250 and NCD224) is also well fit by the appropriate crystallography-based models. However, the measured Rg of hKIN420, 4.05 +/- 0.075 nm, is significantly smaller than that of the crystallography-based model. In addition, the overall scattering pattern of NCD281 is well fit by the model, but that of hKIN420 is poorly fit. Model calculations indicate that the orientation of the catalytic cores is different from that observed in the rKIN379 crystal structure. Like the crystal structure, the best-fitting models do not show 2-fold symmetry about the neck axis; however, their overall shape more resembles a mushroom than the "T"-like orientation of the catalytic cores found in the crystal structure. The center of mass separations of the catalytic cores in the best-fitting models are 0.7-1 nm smaller than in the crystal structure.

Adenosine Triphosphatases↗
PubMed1998

The effect of regulatory Ca2+ on the in situ structures of troponin C and troponin I: a neutron scattering study.

The effects of regulatory amounts of Ca2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied +/-Ca2+ in 41.6% 2H2O buffers in which protonated subunits were rendered "invisible". We found that the radius of gyration (Rg) of TnI decreased by approximately 10% upon addition of regulatory Ca2+ indicating that it was significantly more compact in the presence of Ca2+. The apparent cross-sectional radius of gyration (Rc) of TnI increased by about 9% when regulatory Ca2+ was bound to TnC. Modeling studies showed that the high-Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca2+ addition decreased the center of mass separation by about 1.5 nm. The Rg of TnI, its high-Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca2+) TnC.TnI complex. The Rg of TnC indicated that it was elongate in situ. The Rg of TnC was not sensitive to the Ca2+ occupancy of its regulatory sites. However, Rc increased upon Ca2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca2+-induced changes in TnC.

Animals↗
PubMed1998

Effect of meal dilution on the postprandial glycemic response. Implications for glycemic testing.

OBJECTIVE: To investigate the effect of varying the volume of sugar meals on the post-prandial glycemic response (PGR). RESEARCH DESIGN AND METHODS: On six separate occasions, after an overnight fast, blood glucose concentrations were measured in eight healthy subjects (34 +/- 4 years of age, BMI 22.9 +/- 0.9 kg/m2) after the consumption of 25 g glucose, sucrose, or fructose dissolved in either 200 or 600 ml of water. Blood was obtained at fasting and then at times 15, 30, 45, 60, and 90 min after the start of the test meal. RESULTS: PGR was found to be influenced by carbohydrate type (P < 0.001). Mean response areas (min.mmol.l-1) to the three sugars were statistically different (P < 0.05). Glucose had the highest response area (90.0 +/- 8.1), followed by sucrose (61.3 +/- 5.0) and then fructose (14.7 +/- 2.8). Independent of this effect, PGR was also found to be influenced by volume dose (P < 0.01). By tripling meal volume from 200 to 600 ml, PGR areas were significantly increased for all three sugars, glucose (79.3 +/- 10.3 vs. 100.8 +/- 12.0, P = 0.035), sucrose (52.6 +/- 5.5 vs. 70 +/- 7.4, P = 0.0094), and fructose (11.0 +/- 3.8 vs. 18.4 +/- 3.9, P = 0.012). Where the effects of time (P < 0.05) and dose (P < 0.05) were determined to be independent (interaction nonsignificant) for all three sugars, this increase in volume also significantly increased glycemic concentrations at 15 min, for glucose (P = 0.033) and sucrose (P = 0.026), suggesting that changes in gastric emptying time may be a mechanism of action. CONCLUSIONS: Varying the volume of liquid sugar meals alters PGR. Understanding this concept may help to reduce variability both in the glycemic testing of foods and oral glucose tolerance testing.

Administration, Oral↗
PubMed1996

Conformations of myosin subfragment 1 ATPase intermediates from neutron and X-ray scattering.

In order to elucidate the structural changes that occur during the hydrolysis of ATP by myosin, low-angle neutron and X-ray scattering have been used to investigate the shape of the myosin head (S1) with various bound nucleotides and nucleotide analogs. It was found that the radius of gyration (Rg) of S1.MgADP.AlF4 and of S1MgADP.Vi were similar and significantly smaller (approximately 3%) than the similar Rg values of nucleotide-free S1, S1.MgADP and S1.MgADP.BeFx. In addition, S1 in the presence of MgATP, which is predominantly in the S1.MgADP.Pi state under the experimental conditions employed, showed a change in Rg comparable with that of S1.MgADP.AlF4 and S1.MgADP.Vi. The results obtained here with BeFx and AlF4 are in close harmony with crystallographic results on truncated S1 bearing MgADP.BeFx and MgADP.AlF4. A. Fisher and co-workers have postulated that these two systems, which exhibit some structural differences, represent the pre-hydrolysis state and the transition state of ATP hydrolysis, respectively. It was postulated that this structural difference might alter the orientation of the light-chain-binding domain (tail) of intact S1 relative to the remainder of the molecule. Since this orientation is the major determinant of the Rg of S1, the current data support the hypothesis that a unitary large-scale conformational cocking of S1 for subsequent force production occurs just before or during ATP hydrolysis. Modeling changes in Rg by rigid-body rotations indicates that the longitudinal component of the force-producing throw is likely to be less than 6 nm.

Adenosine Triphosphate↗
PubMed1996

In situ shape and distance measurements in neutron scattering and diffraction.

Neutron scattering combined with selective isotopic labeling and contrast matching is useful for obtaining in situ structural information about a selected particle, or particles, in a macromolecular complex. The observed intensities, however, may be distorted by inter-complex interference and by scattering-length-density fluctuations of the (otherwise) contrast-matched portions. Methods have been proposed to cancel out such distortions (Hoppe's method, the Statistical Labeling Method, and the Triple Isotopic Substitution Method). With these methods as well as related unmixed-sample methods, structural information about the selected particle(s) can be obtained without these distortions. We have generalized these methods so that, in addition to globular particles in solution, they can be applied to in situ structures of systems having underlying symmetry and/or net orientation as well. The information obtainable from such experiments is discussed.

Crystallography↗
PubMed1995

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering.

Reductive methylation of nearly all lysine groups of myosin subfragment-1 (S1) was required for crystallization and solution of its structure at atomic resolution. Possible effects of such methylation on the radius of gyration of chicken skeletal muscle myosin S1 have been investigated by using small-angle neutron scattering. In addition, we have investigated the effect of MgADP.Vi, which is thought to produce an analog of the S1.ADP.Pi state, on the S1 radius of gyration. We find that although methylation of S1, with or without SO42- ion addition, does not significantly alter the structure, addition of ADP plus vanadate does decrease the radius of gyration significantly. The S1 crystal structure predicts a radius of gyration close to that measured here by neutron scattering. These results suggest that the overall shape by crystallography resembles nucleotide-free S1 in solution. In order to estimate the effect of residues missing from the crystal structure, the structure of missing loops was estimated by secondary-structure prediction methods. Calculations using the complete crystal structure show that a simple closure of the nucleotide cleft by a rigid-body torsional rotation of residues (172-180 to 670) around an axis running along the base of the cleft alone does not produce changes as large as seen here and in x-ray scattering results. On the other hand, a rigid body rotation of either the light-chain binding domain (767 to 843 plus light chains) or of a portion of 20-kDa peptide plus this domain (706 to 843 plus light chains) is more readily capable of producing such changes.

Amino Acids↗
PubMed1995

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study.

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.

Animals↗
PubMed1994

The structure of F-actin. Results of global searches using data from electron microscopy and X-ray crystallography.

The structure of F-actin was investigated by fitting the crystallographically determined actin monomer structure to F-actin electron microscopy data sets obtained by a variety of methods. A reciprocal-space global search procedure was applied to non-equatorial reciprocal-space amplitudes and phases of the microscopy data to locate minima. Fits were performed over a range of cross-sectional radii-of-gyration encompassing values obtained from X-ray solution scattering measurements. Five data sets from four laboratories were investigated: one from frozen-hydrated single filaments, three from negatively stained single filaments, and one from negatively stained single-layer paracrystals. In this last case the paracrystal data were straightened to improve resolution. The best fits nearly unanimously favored a monomer orientation having long-pitch connectivity that was close to that obtained by fitting X-ray fiber diffraction patterns. In certain cases where the resolving power was low, competitive fits were obtained with a quite different orientation, one having only protomer connectivity along the genetic helix. Using a running 10-residue deletion from the monomer in the Holmes-type orientation, subtle differences between the monomer structure and the protomer structure in F-actin could be detected. In particular, differences in the "DNase loop" (residues 41 to 50) and the "hydrophobic loop" (residues 264 to 273) were seen in single-filament data. In addition, a perturbation of the structure was seen in a region near residues 81 to 90. A rearrangement within the protomer structure reported in the literature did not produce fits as good as those obtained when using the undistorted monomer crystallographic structure without 10-residue deletions. These results, taken as a whole, provide strong support for a structure of Mg(2+)-ADP F-actin similar to that originally suggested by Holmes et al. but with alterations of the hydrophobic loop, the DNase loop and in the region near residues 81 to 90. These latter two regions have been proposed as secondary binding sites for myosin heads. The available evidence from electron microscopy and from other sources suggests that residues 40 to 49 are disordered in Mg(2+)-ADP F-actin.

Actins↗
PubMed1991

Mechanism of force generation studied by neutron scattering.

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered "invisible" to neutrons when dissolved in 94% D2O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometries, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.

Actins↗
PubMed1991

Recent neutron scattering studies of muscle contraction and its control.

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied. For free chymotrypsin-generated S1 it was found that Rg = 4.0 +/- 0.15 nm, while for papain-generated S1 it was found that Rg = 4.6 +/- 0.2 nm. Upon binding of papain-generated S1 to actin at low NS1/N actin ratios, the change in Rg in difference experiments was delta Rg = 0.05 +/- 0.15 nm. This lack of significant change in Rg in the very low-s domain confirms and extends our earlier neutron scattering work in the higher-s domain. The longest chords of S1, as well as shorter ones, are not significantly altered upon actin binding. These results indicate that muscle contraction does not occur as a result of large-scale changes in S1 structure. In actin-Tm complexes, a measurement of the mean cross-helix separation, d, of Tm molecules has been made using neutron scattering. With deuterated actin matched out in 93% D2O buffer, it was found that d = 7.9 +/- 0.3 nm. This value is in good agreement with a model based on Tm crystallography and also with recent electron microscopy results. These experiments demonstrate the feasibility and value of neutron diffraction and scattering techniques in the study of muscle contraction and its control. One can expect that the further employment of emerging cell biology techniques for generating deuterated proteins will aid our understanding of muscle in the future.

Actins↗
PubMed1991

Cross-helix separation of tropomyosin molecules in acto-tropomyosin as determined by neutron scattering.

The cross-helix separation of Tm molecules in acto-tropomyosin has been determined using neutron scattering. Deuterated Dictyostelium discoideum actin was density matched in a 93% D2O buffer so that effectively only the protonated tropomyosin was "visible" to neutrons. Analysis of the solution scattering pattern in the region of the first oscillation yielded a value for the cross-helix separation of 7.9 +/- 0.3 nm. The implications of this value for the mechanism of the regulation of muscle contraction are discussed in light of recent results by others.

Actins↗
PubMed1988

Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration.

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.

Actins↗
PubMed1984

X-ray scattering by single-headed heavy meromyosin. Cleavage of the myosin head from the rod does not change its shape.

Low angle X-ray scattering from heavy meromyosin (HMM) and from single-headed heavy meromyosin (sHMM) have been examined to determine if the heads of myosin change shape when cleaved from the rod to form subfragment 1 (S1). The scattering intensities of intact HMM and sHMM were compared with those of their chymotryptic digestion products, S1 and subfragment 2 (S2). As the data with HMM were complicated by scattering between the two heads, the more extensive analysis was done with sHMM. Pseudo-Guinier plots of intact and digested sHMM, over the angular range used previously for S1, were linear and showed a difference in apparent radius of gyration (Rg) of only 0.07 +/- 0.04 nm. The absolute apparent Rg value of sHMM was 3.2 +/- 0.2 nm, which is comparable to the radius of gyration reported previously for S1 alone. A plot of the fractional differences in scattering intensities of intact and digested sHMM was flat to a reciprocal spacing of at least 1/3.5 nm-1. These results indicate that the head portions of sHMM and S1 have very similar structures at low resolution. Scattering curves for various models of sHMM and mixtures of S1 and S2 were calculated and the fractional difference plots of scattering intensities were made to determine how sensitive this type of analysis is to changes in the shape of the head. Changes in Rg of 0.1 nm or greater gave detectably non-flat difference plots. Thus, the X-ray scattering of sHMM (and HMM) demonstrated that differences in structure between the head of myosin and isolated S1 are likely to be small. Current controversies over myosin head structure are discussed in light of this result.

Animals↗
PubMed1983

A comparison of order and orientation of crossbridges in rigor and relaxed muscle fibres using fluorescence polarization.

Information has been obtained concerning the spatial disposition of the fluorescent reagent 5-(iodoacetamidoethylaminonaphthalene)-1-sulphonic acid bound covalently to muscle proteins in chemically skinned fibres of rabbit psoas muscle, using a novel time-gated fluorescence detection system to reject scattered incident light selectively. The results are consistent with a model of muscle crossbridge organization in which a particular crossbridge axial angle is strongly favoured in the rigor state. The structure in relaxation is less well ordered, but the favoured axial angle appears to be very close to that in rigor. This conclusion does not depend upon which of the models of crossbridge organization considered here is chosen, and is essentially unchanged if results obtained using a different fluorophore are analysed in the same way.

Animals↗
PubMed1983

Trace mineral balances in preterm infants fed their own mother's milk.

Balance studies were conducted on preterm infants (birthweight, 1,500 g or less) fed their own mother's milk or formula (SMA with iron) to compare the relative adequacy of these sources for copper (Cu), iron (Fe), and zinc (Zn). Urine and stools were collected for 72 h from infants aged 1, 2, or 4 weeks while they were fed milk or formula. Infants fed SMA 24 (but not SMA 20) received Cu, Fe, and Zn intakes within the recommended range. They did not, however, achieve estimated in utero retention rates for Fe, and were in negative Cu balance; they did approach in utero retention rates for Zn. Infants fed their own mother's milk received Cu and Zn, but not Fe, in recommended amounts. They achieved in utero retention rates for Cu at each age studied and approached the estimated in utero retention rates for Zn at 4 weeks, but they retained insufficient Fe throughout. Thus, neither the infant's own mother's milk nor this particular formula provides ideal amounts of all three minerals studied--Cu, Zn, and Fe--during the 1st month of life.

Copper↗
PubMed1982

Zinc, copper and iron content of milk from mothers of preterm and full-term infants.

Complete 24-hour expressions of milk were collected over the first month of lactation from mothers giving birth at term (FT) and prematurely (PT). Samples were analyzed for Cu, Fe and Zn concentration. Composition of PT and FT milks was similar during the first 4 weeks of lactation, but the concentrations of each mineral were higher during the first week than during the fourth week. From these data, the intakes of premature infants fed their own mother's milk were estimated and the proportion which must be absorbed and retained in order to accumulate the amounts laid down in utero were predicted. On the basis of these estimates, preterm infants who retain 25% of the Zn and 35% of the Cu in PT milk would approximate in utero accumulations. However, the Fe content of PT milk is unlikely to provide for in utero accretion rates, even if 100% absorption as achieved.

Adult↗
PubMed1982

Three-dimensional disorder of dipolar probes in a helical array. Application to muscle cross-bridges.

Fluorescence polarization and EPR experiments on azimuthally randomized helices bearing extrinsic (dipolar) probes yield information about the axial orientation and order of the probes. If the orientation of the probe on the structure bearing it is known and disorder is absent, the orientation of the structure may be ascertained. For cases where less probe orientation information is available and/or disorder is present, the available structural information is correspondingly reduced. Here we examine the available data on probes attached to cross-bridges in muscle fibers: four plausible cases of three-dimensional cross-bridge disorders are numerically modeled muscle in states of rigor and relaxation. In rigor, where the reported probe disorder is small (Thomas and Cooke, 1980), it was found that the cross-bridge disorder was also small. On the other hand, for the relaxed state where the probes are found to be completely disordered, the cross-bridges may have a considerable amount of order. This possibility is in concert with the results of x-ray diffraction, in which the presence of well-developed myosin-based layer lines indicates considerable order in relaxed muscle.

Animals↗