Showing papers by "Carlos Bustamante published in 1991"

ORGANIZATION OF PIGMENT‐PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD‐TYPE and CHLOROPHYLL fo‐LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

Abstract: The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.

Direct observation and manipulation of single DNA molecules using fluorescence microscopy.

Abstract: PHYSICAL FOUNDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . 417 Instrumentation and Methods ..... . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Factors Affecting the Image Quality ... . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ . 419 Factors Affecting the Appearance of the Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 The Eff ect of Molecular Confinement in Restricted Geometries. . . . 423 Quantitative Analysis of Systems Containing Few Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

Abstract: A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.

Daunomycin inverts the long-range chirality of DNA condensed states.

Abstract: The effect of daunomycin upon DNA condensed states induced by poly(ethylene glycol) (PEG) was studied by circular dichroism (CD) and circular intensity differential scattering (CIDS). The CD spectra of these aggregates showed psi-type anomalies and intensities 10-100 times greater than those obtained with the dispersed DNA solutions in the absence of PEG. Increasing concentrations of daunomycin, added to the DNA solution prior to its aggregation, led, in the presence of PEG, to CD and CIDS signals which gradually decreased in magnitude and eventually inverted sign. The coincidence of the transition point of both signals and a careful characterization of the CD spectrum at the transition point clearly indicated that the inversion observed corresponds to an inversion of the handedness of the aggregates. The latter result suggests that the structure of the aggregates at the inversion point should resemble that of a nematic liquid-crystalline structure. The characteristic B-DNA spectrum obtained in this case further suggests that the packing process does not affect the secondary structure of the DNA molecules and that small changes in their local structure can induce dramatic changes in their long-range tertiary packing. The results obtained in this study represent a confirmation of a recent theory of psi-type CD in which the anomalous signals are interpreted as a manifestation of the long-range chirality of the aggregates.

A method for imaging E. coli. RNA polymerase holoenzyme with the scanning tunneling microscope in an aqueous environment

Abstract: An electodeposition method has been developed for depositing E. coli. RNA polymerase onto a monoatomically flat gold surface. The scanning tunneling microscope has been used to image this enzyme in a high‐humidity or water/glycerol environment, which allows direct observation without metal coating. The deposition technique is reproducible enough to allow optimization of the deposition parameters for controlled electrodeposition. Time‐dependence studies were conducted to help determine the binding mechanism of the protein. The enzyme molecules appear as ordered arrays, amorphous features, or ‘‘jaw‐shaped’’ molecules depending on the deposition conditions used. The ‘‘jaw‐shaped’’ molecules closely resemble those observed by electron microscopy. In some of these a groove 22 A wide can be seen.

Manipulation of single-DNA molecules and measurements of their elastic properties under an optical microscope

Abstract: Single molecules of DNA can be visualized in solution by epifluorescence microscopy, manipulated and extended by a variety of mechanical, electrical and chemical means as described previously. This has been used to design experiments under an optical microscope, in which DNA molecules are extended by a known force, to determine the elastic response of the molecules, both in the presence and absence of ethidium bromide. It is found that at lower forces (smaller extensions) the molecules behave as entropic springs with a persistence length of 500 angstroms, and that at the ionic strengths used, the intercalation of ethidium bromide does not alter this persistence length, while it appears to elongate the contour length of the molecule by about 30%.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Circular intensity differential scattering measurements in the soft x-ray region of the spectrum (~16 EV to 500 EV)

Abstract: We propose the use of recently developed technique of circular intensity differential scattering (CIDS), as extended to the soft x-ray region of the spectrum (16 eV to 500 eV), to study the higher order organization of the eukaryotic chromosome. CIDS is the difference in scattering power of an object when illuminated by right circularly polarized vs. left circularly polarized electromagnetic radiation of arbitrary wavelength. CIDS has been shown to be a very sensitive measure of the helical organization of the scattering object, e.g., the eukaryotic chromosome. Preliminary results of measurements of samples of bacteriophages and octopus sperm done at SRC, Wisconsin, show the technique to be very sensitive to the dimensional parameters of the particles interrogated by circularly polarized light.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.