Kinetics and thermodynamics of phenotype: unwinding and rewinding the nucleosome.

Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo.

A new bright green-emitting fluorescent protein--engineered monomeric and dimeric forms.

Fluorescent proteins have become essential tools in molecular and biological applications. Here, we present a novel fluorescent protein isolated from warm water coral, Cyphastrea microphthalma. The protein, which we named vivid Verde fluorescent protein (VFP), matures readily at 37 degrees C and emits bright green light.

Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded).

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins.

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions.

Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures.

Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment-protein interactions responsible for the positions of the Qy bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Qy and Soret transition energies.

Femtosecond time-resolved absorption spectroscopy of main-form and high-salt peridinin-chlorophyll a-proteins at low temperatures.

Steady-state and femtosecond time-resolved optical methods have been used to compare the spectroscopic features and energy transfer dynamics of two systematically different light-harvesting complexes from the dinoflagellate Amphidinium carterae: main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a-proteins. Pigment analysis and X-ray diffraction structure determinations [Hofmann, E., Wrench, P.

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers.

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules.

Femtosecond time-resolved absorption spectroscopy of astaxanthin in solution and in alpha-crustacyanin.

Steady-state absorption and femtosecond time-resolved spectroscopic studies have been carried out on astaxanthin dissolved in CS2, methanol, and acetonitrile, and in purified alpha-crustacyanin. The spectra of the S0 --> S2 and S1 --> S(n) transitions were found to be similarly dependent on solvent environment. The dynamics of the excited-state decay processes were analyzed with both single wavelength and global fitting procedures.

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae.

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P.