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Investigates the purification and crystallization of RNA polymerase (RNAP) holoenzyme from Thermus thermophilus. Molecular mechanism of RNAP; Structure of RNAP core enzyme; Process for holoenzyme purification of T. thermophilus cells.

A new system for thermoluminescence (TL) and delayed luminescence (DL) measurements with precise temperature control was developed. A freezing/heating stage for microscope was modified to afford a fixed sample temperature and a constant heating rate. A valve equipped with a low-flow bypass for cold N [ABSTRACT FROM AUTHOR]

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 Å resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA. [ABSTRACT FROM AUTHOR]

The S2 state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S2 forms. FTIR difference spectra for S2/S1 as well as for S2QA -/S1QA measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S2 formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S2 state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S2 forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands. [ABSTRACT FROM AUTHOR]

Discusses the structure of histidine ligands in the photosynthetic oxygen-evolving complex examined through light-induced fourier transform infrared difference spectroscopy. Histidine side chain model compounds and effect of N-deuteration; Hydrogen bonding in the N-H stretching bands.

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni2+-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 μmol (mg Chl)−1 h−1 at 45°C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2−, B1− and Q-bands arising from S2QB−, S3QB− and S2QB− charge recombinations at respective emission temperatures of 45, 38 and 20°C, all of which were higher by about 15°C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20°C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII. [ABSTRACT FROM AUTHOR]

Investigates the structure and molecular interactions of the primary donor in the reaction center of photosystem II by detecting light-induced FT-IR difference spectra upon the formation of the triplet states of purified chlorophyll. FT-IR difference spectrum of P680/P680 in PS II RC; FT-IR difference spectra of T1/s0 in purified Chl; More.

Presents information on a study which focused on the interactions of the primary quinone acceptor QA of photosystem II with surrounding amino acid residues. Materials and methods; Results and discussion.

Presents a study that attempts to measure the flash-induced Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon S1-to-S2 transition, through the use of photosystem II core complexes of Synechocystits. Methodology utilized in the study; Results and conclusions of the study.

The limited proteolysis of the Bacillus stearothermophilus pyruvate dehydrogenase complex by V8 protease yields its core structure solely composed of lipoate acetyltransferase (E2) fragments. The changes in the core with guanidine hydrochloride (GdnHCI) were biphasic: below 0.8 M (first) and above 1.0 M (second) GdnHCI. The changes in the first phase were slight but significant: decreases in ellipticity and light scattering, and an increase in E2 activity. Insignificant changes in the molecular shape and size of the. core were detected on fluorescence spectroscopy, ultracentrifugation, gel filtration, and electron microscopy. On the other hand, the changes in the second phase were drastic; the core was disassembled and denatured. [ABSTRACT FROM AUTHOR]