and Aileen K. W. Taguchi

Development of a fluorescent probe for the study of nucleosome assembly and dynamics.

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.

Mutations that Affect the Donor Midpoint Potential in Reaction Centers from Rhodobacter Sphaeroides

Modulation of the redox midpoint potential of the initial electron donor is key for achieving electron transfer with high yields in photosynthetic systems. The initial electron donor in reaction centers of purple nonsulfur bacteria, P, has a much lower potential than the donor in photosystem II reaction centers, which are capable of oxidizing water (for general reviews of chlorophylls and their oxidation potentials, see ref. 1). Isolated bacteriochlorophyll and chlorophyll have similar oxidation potentials of approximately 700 mV and 800 mV, respectively. However, in vivo the midpoint oxidation potential of the donor in bacterial systems, approximately 490 mV, is much lower than that of monomer bacteriochlorophyll, while that of the donor in photosystem II is higher than 1.0 V. This dramatic alteration of the oxidation potentials in the reaction center must be due to interactions between chlorophylls or between the donor and the surrounding protein. This is supported by the observation of changes of approximately 100 mV in the oxidation potentials for bacteriochlorophylls and chlorophylls due to changes in solvent1. In this paper we address the role of specific protein-donor interactions in modulating the potential.

FTIR Characterization of Leu M160→His, Leu L131→His and His L168→Phe Mutations Near the Primary Electron Donor in RB. Sphaeroides Reaction Centers

Light-induced FTIR difference spectroscopy has been used to monitor structural changes in Rb. sphaeroides mutant reaction centers (RCs) associated with the substitution of amino acids near the primary electron donor (P). In the wild type Rb. sphaeroides RC, the 9keto carbonyls for both BChls constituting P (PL and PM) have no specific interactions with the protein. A hydrogen bond probably exists between the 2a acetyl C=O of PL and His L1681,2 but no such bond is possible with the symmetry related amino acid on the M side, a Phe residue (M197). The mutations Leu L131→His and Leu M160→His3 (see also Williams et al., these proceedings) were designed to introduce a proton donating residue that could form a hydrogen bond with the keto C=O of ring V of each BChl of the dimer. In addition, the mutation His L168→Phe was designed to break a hydrogen bond between the 2a C=O of PL and His L168.