Steady-state and transient electric birefringence of solutions of bent-rod macromolecules

We first calculate the steady-state birefringence, expressed in the form of specific Kerr constant, K_sp, of rigid, bent-rod macromolecules. Equations are derived for K_sp as a function of the geometric and electro-optical parameters. We also consider flexibly hinged rods and evaluate K_sp for them by averaging over the angle between the two arms, ?. Next, we turn to the time decay of the electric birefringence. The decay function for rigid bent rods is a sum of three exponential terms, and a procedure for their calculation is indicated. We observe that single-exponential decays can be found for ? > 90° or ? < 60°, in spite of the high electro-optical and hydrodynamic anisotropy of the macromolecule. Special attention is paid to the case of rods with equal arms.     

Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production.     

Ultra sensitive sensor with enhanced dynamic range for high speed detection of multi-color fluorescence radiation

This paper describes design of the new ultra sensitive sensor system for fluorescence detection applications. System comprises two units: optical spectra separation unit and detection unit. Optical unit of the sensor performs spatial spectra separation of signal from the laser excited fluorescence, and resulting spectra is collected in the detection part of the system. Optical part is built using diffraction grating as spectra separation element. Detection part comprises 32-channel photomultiplier tube working in single photon counting mode with our 32-channel amplifier. Using single photon detection technique and specific signal processing algorithms for collected data, the proposed system allows to achieve unique combination of characteristics--high sensitivity, high detection speed and wide linearity dynamic range comparing to existing commercial instruments. DNA sequencing experiments with new sensor as detection device, and using two types of lasers (Ar-ion and Nd-YAG) were carried out, yielding sequencing traces which have quality factor of 20 for read lengths as long as 650 base pairs.     

Analysis of DNA bending by transient electric birefringence

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989-995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30 degrees to 41 degrees were calculated for the target fragments by tau-ratio method. The bend angles of four of the target fragments were independent of temperature from 4 degrees C to 20 degrees C, but decreased when the temperature was increased to 37 degrees C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4 degrees -37 degrees C. Hence, the thermal stability of sequence-dependent bends in random-sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the tau-ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence-dependent bending in other random-sequence DNAs.     

A simple method for the extraction of soil solution by high speed centrifugation

Summary A simple technique for extracting samples of soil solution by high speed centrifugation is described. The method uses inexpensive commerically available centrifuge tubes requiring only slight modification. Aspects of the method are discussed together with an example of its application.     

Electric properties of macromolecules. IX. Dipole moment, polarizability, and optical anisotropy factor of collagen in solution from electric birefringence

The electric birefringence of collagen solutions has been measured over a wide range of field strength with the pulse technique. The soluble collagen was from rat tail tendon. The solvent used was dilute acetic acid. Very pronounced saturation of the electric birefringence was observed, permitting calculation of the optical anisotropy factor. The Kerr constant was determined by extrapolation to zero field strength. From the dependence on field strength of the birefringence, the permanent dipole moment and the anisotropy of polarizability were separately determined. The contribution of the former to the Kerr constant was found to be twice as large as that of the latter. The same conclusion was obtained from the initial slope of the rise curves of the birefringence at low fields. The permanent dipole moment was 1.5 × 10⁴ Debye, and the anisotropy of polarizability was about 3 × 10^?15 cm.³. The magnitude of the latter indicates that the ion atmosphere polarization is important. Effects of added salt and thermal denaturation on the electric birefringence were explored.     

X-ray diffraction studies of the conformation of macromolecules in solution. 3. The helix-coil transition

The procedure outlined in the previous paper of this series is applied to x-ray diffraction patterns of labeled polytyrosine in dimethylformamide–trifluoroacetic acid solutions. The α-helix appears to transform into the random coil conformation at a trifluoroacetic acid concentration between 40 and 42.5%. Comparison of these results with those measured in the same system by optical rotation are in essential agreement.     

Investigation of the flexibility of DNA using transient electric birefringence

In the preceding article, a Monte Carlo analysis was presented which provides a quantitative numerical relationship between the rotational diffusion coefficients, as measured by the decay of optical anisotropy following an electric field pulse, and the flexibility (persistence length) of short, wormlike chains. In the present article, the results of the foregoing analysis are applied to the observed rates of decay of birefringence for a series of sequenced DNA fragments ranging in size from 104 to 910 base pairs. Under the conditions used in this study, the DNA fragments exist as native, duplex molecules. Furthermore, conditions are defined in which the observed relaxation times are not dependent on DNA concentration, field strength, or the duration of the pulse. It is pointed out that the ionic atmosphere associated with a wormlike polyion does not exert any significant (direct) influence on the rotational diffusion of the polyion and, therefore, that the rotational relaxation times are a true measure of the configurations of the DNA molecules in solution. Moreover, excluded-volume effects are shown not to be significant for the moderately short molecules employed in this study. The major conclusion of this study is that there is no strong ionic strength dependence of the persistence length for ionic strengths above 1 mM and that the persistence length, under conditions where electrostatic contributions are negligible, is approximately 500 Å. For ionic strengths significantly lower than 1 mM, electrostatic contributions to the stiffness of DNA become significant.     

Electric birefringence of ionized polypeptides in solution and the effect of high electric fields on the helix-coil transition

Electric birefringence and circular dichroism measurements have been made on solutions of two po!y (L-lysine) homologs. The specific Kerr constant and the molar ellipticity at 222 nm of poly (L-alpha, gamma-diaminobutyric acid hydrochloride) in methanol/water mixtures underwent an abrupt change between 75 and 80 vol% methanol at 25 degrees C, corresponding to a solvent-induced helix-coil transition. On the helix side of the transition region, i.e., between 78 and 80 vol% methanol, anomalous birefringence transients indicative of field-induced helix-to-coil transition were observed at high fields. In the case of poly (L-ornithine hydrobromide) in methanol/water mixtures, a helix-coil transition was induced between 93 and 98 vol% methanol and anomalous birefringence transients were observed between 96 and 98 vol% methanol. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for various solvent compositions and polymer concentrations could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transients were observed. This enabled us to estimate the threshold field strength.     

Computer simulation studies of self-assembling macromolecules

Coarse-grained (CG) molecular models are now widely used to understand the structure and functionality of macromolecular self-assembling systems. In the last few years, significant efforts have been devoted to construct quantitative CG models based on data from molecular dynamics (MD) simulations with more detailed all-atom (AA) intermolecular force fields as well as experimental thermodynamic data. We review some of the recent progress pertaining to the MD simulation of self-assembling macromolecular systems, using as illustrations the application of CG models to probe surfactant and lipid self-assembly including liposome and dendrimersome formation as well as the interaction of biomembranes with nanoparticles.     

A study of rhodopsin-detergent micelles by transient electric birefringence.

The hydrodynamic method of transient electric birefringence has been used to study bovine rhodopsin solubilized in two detergents, 0.02% Ammonyx LO and 0.045% digitonin. All measurements are interpreted as the sum of two exponentials by which the relaxation times yield the rotary diffusion coefficients for ellipsoids of revolution. The semi-major and minor axes for prolate ellipsoid models have been calculated and their axial ratio, 6.8, in both detergents, is in line with recent reports on the structure of rhodopsin. Studies on bleached rhodopsin showed a large increase in axial ratio in 0.02% Ammonyx LO.     

Deoxygenation of solutions for transient studies

An all-glass vessel which allows a solution to be freed of dissolved oxygen was constructed. The main advantage of this apparatus is the ability to deoxygenate the system without the addition of a reducing agent. This might be desirable in order to avoid possible interactions of highly reactive systems or excited states with foreign additives. Other advantages of the method over the customary freeze-pump-thaw method or vigorous bubbling by an inert gas are (i) the ability to deoxygenate solutions of sensitive biological macromolecules by gentle means; (ii) the ability to add aliquots of reagents to a solution without exposing it to the ambient atmosphere; and (iii) the all-glass vessel's leak-proof quality toward the atmosphere for hours after freeing the solution from oxygen. The apparatus should be useful for spectroscopic, i.e., absorption and emission, studies. It was tested by measuring the 10-ms triplet lifetime of Zn-protoporphyrin which was substituted for the native Fe-porphyrin in myoglobin and the 5-ms lifetime of Zn-octaethylporphyrin in toluene.