Subunit Rotation Research Articles

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.

X-Ray studies reveal lanthanide binding sites at the A/B 5 interface of E. coli heat labile enterotoxin

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.

Microwave spectrum, dipole moment, structure, and internal rotation of the cyclopropane-sulfur dioxide van der Waals complex

The rotational spectrum of the cyclopropane-sulfur dioxide complex was observed by Fourier transform microwave spectroscopy. The spectrum exhibited a- and c-dipole selection rules with the c-dipole transitions split into doublets of unequal intensity separated by about 150 kHz. The structure has Cs symmetry with the sulfur and carbon atoms all lying in the ac plane; the oxygen and hydrogen atoms straddle the plane. The sulfur dioxide plane is nearly parallel to a C–C bond edge. The distance from the center of mass of the SO2 to the C–C bond center is 3.295 Å. The dipole moment of the complex is 1.681(1) D, with components μa=0.815(1) D and μc=1.470(1) D. The splittings in the spectrum arise from an internal rotation of the cyclopropane subunit about its local C2 axis which lies nearly along the line connecting the centers of mass.

Slit-jet near-infrared spectroscopy and internal rotor dynamics of the ArH2O van der Waals complex: An angular potential-energy surface for internal H2O rotation

Near-infrared vibration-rotation spectra of jet-cooled Ar-H2O complexes are detected for the first time via direct absorption of tunable difference frequency infrared radiation in a slit supersonic expansion source. Transitions from both the lowest para and ortho complexes are observed which correlate to 000 and 101 rotational levels of free H2O, respectively, and permit spectroscopic characterization of the complex in both the ground (ν3=0) and asymmetric stretch excited (ν3=1) levels. From hot bands involving excited internal rotor states correlating with the 101 H2O rotational level, we determine the Σe(101)-Πf(101) energy splitting to be 11.3333(3) cm−1 (J=1). In conjunction with far-infrared measurements of the Σ(110)-Π(101) and Π(110)-Σ(101) energy splittings, this information permits determination of a two-dimensional (2D) angular potential-energy surface of the complex as a function of the H2O orientation. The barriers to internal rotation of an ArH2O differ for in-plane (19 cm−1) and out-of-plane (33 cm−1 ) rotation of the H2O subunit. However, both barriers are only slightly higher than the ground-state energies and, hence, the behavior of H2O in the complex is that of a nearly free rotor. Agreement with recent ab initio calculations by Chalasinski et al. [J. Chem. Phys. 94, 2807 (1991)], as well as with 3D fits solely to far-infrared data by Cohen and Saykally [J. Chem. Phys. 94, 7991 (1991)], is remarkably good. Predictions based on this angular potential-energy surface are made for the internal rotor states of ArHDO and ArD2O and compared with recent far-infrared measurements by Suzuki et al. [J. Phys. Chem. 94, 824 (1991)].

Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite I.

The resolvase-catalysed reaction between two res sites in a circular DNA substrate normally gives two circular recombination products linked in a two-noded catenane. Homology between the two res sites at the central overlap dinucleotide of subsite I is important for recombination. Reactions between res sites differing at one position in the central dinucleotide (AC X AT) gave a low yield of recombinants containing mismatched base-pairs, but gave large amounts of a non-recombinant four-noded knot. This result was predicted by a 'simple rotation' model for strand exchange. The mismatch is evidently recognized only after commitment to an initial 180 degrees rotation of the resolvase-linked DNA ends, and it induces a second 180 degrees rotation which restores correct base-pairing at the overlap, giving the four-noded product. Correct base-pairing is not essential for religation, but may be important for release of the products. Characteristic patterns of 4, 6, 8 and 10 node knots, or 4, 8, 12 and 16 node knots were obtained, depending on the reaction conditions and the resolvase. Two pathways for multiple rounds of rotation in 360 degrees steps are inferred. The results support a model for strand exchange by supercoil-directed subunit rotation within a resolvase tetramer.

Studies on DNA-cleaving agents: computer modeling analysis of the mechanism of activation and cleavage of dynemicin-oligonucleotide complexes.

Dynemicin A is a recently identified antitumor antibiotic. Upon activation, dynemicin is reported to cause double-stranded cleavage of DNA, putatively through the intermediacy of a diradical. Computer modeling of this activation and cleavage process is described herein as part of an effort to establish a structural hypothesis for this mechanistic sequence and for the design of simple analogues. Intercalation complexes of duplex dodecamers [d(CGCGAATTCGCG)]2 and [d(GC)6]2 with both enantiomers of dynemicin and of all related mechanistic intermediates are evaluated. Examination of these structures shows that cycloaromatization of dynemicin to a diradical intermediate results in the rotation of the diradical-forming subunit with respect to the intercalation plane that is of an opposite sense for the two dynemicin enantiomers. In addition, the activation of the (2S) enantiomer of dynemicin occurs by a less restricted approach trajectory than the corresponding (2R) enantiomer. In all complexes, the 5'-3' strand is at least 1 A closer than the 3'-5' strand to the diyl intermediate. As a result, complexes are produced in which the diyl moiety is aligned along [(2S)] or across [(2R)] the minor groove, leading to different predictions for the selectivity of radical-initiated, oxidative lesion of DNA. Molecular dynamics simulations are found to support these predictions, including the 3-base-pair offset cleavage reported for dynemicin.

Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots.

The Hin recombinase of Salmonella normally catalyzes a site-specific DNA inversion reaction that is very efficient when the Fis protein and a recombinational enhancer sequence are present. The mechanism of this recombination reaction has been investigated by analyzing the formation and structure of knots generated in different plasmid substrates in vitro. Hin seldom knots the wild-type substrate under standard recombination conditions. However, we show that increasing the length of DNA between the recombination sites and the enhancer and changing the sequence of the core nucleotides where strand exchange occurs increases the efficiency of the knotting reaction. The structure of the knots generated by different mutant substrates strongly supports a model involving a unique configuration of DNA strands at synapsis and DNA strand exchange mediated by rotation of one set of Hin subunits after DNA cleavage. Analysis of the stereostructure of the knots by electron microscopy of RecA-coated DNA molecules demonstrates that the direction of subunit rotation is exclusively clockwise. Because multiple subunit rotations generating knotted molecules do not occur efficiently when the enhancer is located in its native position, we suggest that the enhancer normally remains associated with the two recombination sites in the invertasome structure during strand exchange to limit strand rotation once it has been initiated. Under certain conditions, however, complex knots are formed that are probably the result of the premature release of the enhancer and multiple, unrestrained subunit exchanges.

A molecular switch: Subunit rotations involved in the right-handed to left-handed transitions of Salmonella typhimurium flagellar filaments

Using the combined techniques of cryoelectron microscopy and image analysis, we generated three-dimensional reconstructions of flagellar filaments from straight, right-handed (SJW1655-R) and straight, left-handed (SJW1660-L) Salmonella typhimurium mutants, both of which have the same parental strain (SJW1103). In the filaments from SJW1655, all flagellin subunits have the same conformation (R), while in filaments from SJW1660, the subunits are all in the alternate (L) conformation. The difference between the two three-dimensional density maps reveal the structural changes that accompany switching of the flagellin subunits between the two conformations. In going from the R to L state, the subunit undergoes a rotation 30 degrees clockwise about a radial axis and 38 degrees clockwise about a vertical axis, and suffers a 50 degrees bend of the outer, relative to the inner, subunit domain. The intersubunit spacing, along the 11-start protofilaments, changes from 51.6 A in the right-handed filament to 52.1 A in the left-handed filament. In order to produce the correct corkscrew shape in native filaments, the change in contacts that produces this shortening of 0.5 A must occur among the inner domains at a radius of about 30 A. We suggest that the changes in the middle domains of the subunit are the switch that forces changes in the inner domains.

Is the carbon monoxide—acetylene dimer semirigid? Evidence from its rotational spectrum

The ground-state rotational spectra of ten isotopomers of the carbon monoxide—acetylene dimer are reported and the spectroscopic constants B O and D J determined in the usual way in a semirigid rotor fit for a linear molecule. The B O values are consistent with a simple linear model of the dimer with nuclei in the order OC…HCCH. The failure to observe the isotopomer OC…HCCD in mixtures of CO and HCCD is interpreted in terms of a zero-point energy difference rather than in terms of a nearly free internal rotation of the acetylene subunit.

A microwave spectral and ab initio investigation of O 3H 2O

Microwave spectra of O 3H 2O, O 3H 2 18O, O 3HDO, and O 3D 2O have been observed with a pulsed-beam Fabry-Perot cavity Fourier-transform microwave spectrometer. Two tunneling states, designated A 1 and A 2, are found for the normal and dideuterated isotopic forms, while only one state is observed for the O 3HDO isotope. The A 1 spectra of O 3H 2O, O 3H 2 18O, and O 3D 2O as well as the O 3HDO spectrum were fit to a Watson asymmetric top Hamiltonian, giving A = 11 960.584(5), B = 4174.036(8), and C = 3265.173(8) in MHz for O 3H 2O. The A 2 states of O 3H 2O and O 3D 2O could not be fit to a Watson Hamiltonian. This result, when combined with the large frequency splittings and the observed nuclear spin statistics for the O 3H 2O and O 3D 2O isotopic forms, suggests that there is a low barrier to internal rotation of water. The tunneling motion may also involve ozone through a concerted internal rotation of both monomer subunits. Stark effect measurements of a- and c-type transitions for O 3H 2O give the electric dipole components μ a = 1.014(2) D and μ c = 0.522(6) D. The dipole and moment of inertia data indicate that the complex has C s symmetry with water and the unique oxygen of ozone lying in the symmetry plane. This plane bisects the OOO angle of ozone. The distance between the centers of mass of ozone and water is 2.957(2) Å. From the microwave data and ab initio calculations at the MP2 6–31 G (d, p) and MP4( SDTQ) 6–31 G (d, p) level of theory it is found that the terminal oxygens of ozone are tilted toward one of the nonequivalent hydrogen atoms in water. Furthermore, the calculations reveal that O 3 and H 2O adopt an orientation within the complex that guarantees maximal stabilization by electric dipole-dipole attraction.

Microwave spectrum, structure, barrier to internal rotation, dipole moment, and deuterium quadupole coupling constants of the ethylene–sulfur dioxide complex

The microwave spectra of the complex between ethylene and sulfur dioxide and nine of its isotopic species have been observed in a Fourier transform microwave spectrometer. The spectra exhibit a and c dipole selection rules; transitions of the normal species and several of the isotopically substituted species occur as tunneling doublets. The complex has a stacked structure with Cs symmetry; the C2H4 and SO2 moieties both straddle the mirror plane with the C2 axis of SO2 crossed at 90 ° to the carbon–carbon bond axis (i.e., only the S atom lies in the symmetry plane). The distance between the centers of mass (Rcm) of C2H4 and SO2 is 3.504(1) Å and the deviation of their planes from perpendicular to Rcm is 21(2) ° and 12(2) °, respectively. The tunneling splittings arise from a rotation of the ethylene subunit in its molecular plane. The barrier to internal rotation is 30(2) cm−1. The dipole moment of the complex is 1.650(3)D. The deuterium nuclear quadrupole coupling constants for C2H3D⋅SO2 are χaa=−0.119(1) MHz, χbb=0.010(1) MHz, and χcc=0.109(1) MHz. The binding energy is estimated to be 490 cm−1 from the pseudo-diatomic approximation. A distributed multipole electrostatic model is explored to rationalize the structure and binding energies.

The near-infrared spectrum of NeHCl

The near-infrared spectrum of jet-cooled NeHCl is reported. The HCl stretching fundamental and three combination bands are observed and analyzed to determine rotational constants and vibrational frequencies including those of the van der Waals stretching and bending modes. An unusual intensity distribution is observed, in which the fundamental is weak and the combination bands are strong; in part this is the result of nearly free internal rotation of the HCl subunit within the complex.

Changements cytochimiques et ultrastructuraux des parois cellulaires de la pellicule du raisin, Vitis vinifera, durant la croissance et la maturation de la baie

Cytochemical and ultrastructural analysis of cell wall characteristics was performed in the dermal system of the grape berry (Vitis vinifera L. cv. Pinot noir), using a test for polysaccharides (periodic acid – thiosemicarbazide – silver proteinate, associated with dimethylsulfoxide extraction). Samples were examined at successive stages of fruit development. The main events of the first growth phase, i.e., thickening of hypodermal cell walls and their tangential extension, continued during the second growth phase. At all the stages of grape development, tangentially expanding walls were typical of actively growing plant cell walls, with an ordered texture, new layer deposition, and progressive rotation of microfibrillar subunits (bow-shaped patterns). At maturity, hydration, swelling of the walls, and an increase in the amount of soluble pectic substances were noticed. No redifferentiation of the cell walls occurred. The cuticle, already differentiated into two layers at the first growth phase, was clearly lamellar during the second. At maturity a number of layers, distinguishable by their cytochemical reactivity, were detected. Cuticle thickness increased during "véraison" and was maintained throughout the second growth phase. A considerable increase in the content of epicuticular waxes was noticed during ripening.

Van der Waals modes of solute/solvent clusters: benzene-methane, -deuteriomethane, and -carbon tetrafluoride

Clusters of benzene(CD/sub 4/)/sub 1/ and -(CF/sub 4/)/sub 1/ are created in a supersonic molecular jet and are studied by two-color time-of-flight mass spectroscopy. The clusters' S/sub 1/ reverse arrow S/sub 0/ intermolecular vibronic structures are characterized by calculational modeling of the clusters' intermolecular motion. The calculations include (1) an intermolecular normal-coordinate analysis (NCA) which treats all six van der Waals modes under a harmonic oscillator assumption and (2) a three-dimensional hindered rigid-rotor analysis (3D-HRRA) which treats the intermolecular torsional motion. Agreement between calculation and experiment is excellent for binding energy, symmetries, and van der Waals mode energies. The cluster spectra and calculated intermolecular modes are compared to those of benzene(CH/sub 4/)/sub 1/ reported previously. A major conclusion of this work is that the clusters behave rigidly with regard to internal rotation of the cluster subunits and that the clusters possess unique equilibrium geometries. The internal torsional motion is oscillatory and is constrained by an orientationally dependent intermolecular potential whose barrier height is of the order of the cluster binding energy.

Rotational spectrum and structure of CF3H–NH3

The rotational spectrum of CF3H–NH3 has been obtained using a pulsed nozzle Fourier transform microwave spectrometer. A symmetric top spectrum is observed that is consistent with free internal rotation of the NH3 subunit against the CF3H subunit. Rotational transitions have been measured for both the ground and first excited internal rotor state of the complex. The spectroscopic constants which have been obtained include: B0=1996.903(2) MHz, DJ =3.46(12) kHz, and eQqN =−3.186(8) MHz. From the quadrupole coupling constant of the nitrogen nucleus, eQqN, the bending amplitude of the NH3 unit is determined to be 22.57(10)°. The hydrogen bond length is 2.314(5) Å and the weak bond stretching force constant is 0.066(2) mdyn/Å. The bond length and stretching force constant for CF3H–NH3 are similar in value to those determined for HCCH–NH3 (2.33 Å and 0.070 mdyn/Å, respectively).

The rotational spectra of NH3–CO and NH3–N2

The rotational spectra of NH3–CO, ND3–CO, ND2H–CO, NDH2–CO, NH3–13CO, and NH3–N2 have been measured by molecular beam electric resonance. The K=0 ground vibrational state transitions for these species were fit to a linear molecule Hamiltonian and the following constants were obtained for NH3–CO; (B+C)/2 (MHz)=3485.757(2), DJ (kHz)=110.2(2), eQqNaa (MHz)=−1.890(7), μa (D)=1.2477(8). These constants were also determined for ND3–CO [3078.440(7), 75.7(8), −2.028(15), 1.2845(9)], NHD2–CO [3202.303(4), 86.8(6), −1.972(11), 1.2686(8)], NH2D–CO [3338.235(4), 98.9(6), −1.916(12), 1.2546(8)], NH3–13CO [3451.684(5), 108.7(7), −1.870(15), 1.2452(8)]. For NH3–N2 (B+C)/2=3385.76(21), DJ =117.(10), and μa =1.069(14). For NH3–CO three ‖ΔJ‖=1, K=0 progressions were seen along with two ‖ΔJ‖=1, K=1 progressions, suggesting nonrigidity in the complex. The internal rotation of the NH3 subunit about its C3 axis is expected to be essentially free, but this motion, by itself, is not sufficient to explain the observed spectra, thus, large amplitude dynamics are occurring in at least two degrees of freedom. The quadrupole coupling constants, eQqNaa indicate that in each of the isotopes of NH3–CO the NH3 subunit has its C3 axis relatively rigidly oriented at an angle of approximately 36° with respect to the line connecting the centers of mass of the two subunits. The structure is not hydrogen bonded; the N atom is closest to the CO subunit. The orientation of the CO subunit is not established. The distance between the N atom and the center of mass of the CO unit (RN–CO) is 3.54(3) Å. The spectroscopic constants suggest that the weak bond stretching force constant is quite small (0.01 mdyn/Å) but compatible with the long bond length.

Transmembrane macromolecule rotation model - Molecular hypothesis of membrane excitation.

A hypothesis concerning the molecular changes in the nerve cell membrane during excitation is presented. Based upon established principles about the interaction of hydrophobic and hydrophilic ligands and upon recent facts concerning the contraction of neurofilaments, the hypothesis accounts for both the initiation of the action potential and its conduction along the nerve axon without attenuation. The hypothesis can be summarized as follows: When acetylcholine combines with the acetylcholine receptor (AchR) alpha subunit, hydrophobic subsites are exposed at the bound surface of AchR. Since hydrophobic portions are quite unstable, the subsites move to the center of the membrane, which is hydrophobic. It is postulated that the rotation of receptor subunits makes the center an ion channel. Ca++ flowing in through the ion channel causes the undercoating filaments to contract and this contraction results in the conduction of an action potential along the axon. The contraction of the undercoating filaments induces transmembrane rotation of globular protein connected to the filaments. At right angles to the axon the force of the hydrophobic bond between membrane macromolecules is so strong that many cylindrical lipid structures are formed. The gaps between the rotatory cylinders serve as ion channels. Ca++ passing through the ion channels allows for the conduction of an action potential without attenuation.

Investigation, by cross-linking, of conformational changes in F-actin during its interactions with myosin.

The hypothesis that the subunits of F-actin rotate during interactin with myosin and ATP has been tested by using the specific cross-linking reagent p-phenylene-N,N'-bis(maleimide) (PM). The insertion of cross-links between F-actin subunits does not change the ability of the F-actin to activate the ATPase of either myosin subfragment-1 (S-1) or heavy meormyosin, and its ability to superprecipitate with myosin is unimpaired. We conclude that large-scale rotations of actin subunits are not required for activity. The cross-linking of F-actin by PM is, however, inhibited in a noncooperative fashion by S-1 binding, suggesting that a small local change in actin structure may accompany the binding of S-1 or that S-1 sterically blocks the cross-linking by binding near the contact region between actin subunits.

Fluid Mechanical Aspects of Antisymmetric Stress

Basic fluid mechanical concepts are reformulated in order to account for some structural aspects of fluid flow. A continuous spin field is assigned to the rotation or spin of molecular subunits. The interaction of internal spin with fluid flow is described by antisymmetric stress while couple stress accounts for viscous transport of internal angular momentum. With constitutive relations appropriate to a linear, isotropic fluid we obtain generalized Navier-Stokes equations for the velocity and spin fields. Physical arguments are advanced in support of several alternative boundary conditions for the spin field. From this mathematical apparatus we obtain formulas that explicitly exhibit the effects of molecular structure upon fluid flow. The interactions of polar fluids with electric fields are described by a body-torque density. The special case of a rapidly rotating electric field is examined in detail and the induction of fluid flow discussed. The effect of a rotating electric field upon an ionic solution is analyzed in terms of microscopically orbiting ions. This model demonstrates how antisymmetric stress and body torque can arise in ``structureless'' fluids.