Molecular organization of histidine-tagged biomolecules at self-assembled lipid interfaces using a novel class of chelator lipids.

In molecular biology, the expression of fusion proteins is a very useful and well-established technique for the identification and one-step purification of gene products. Even a short fused sequence of five or six histidines enables proteins to bind to an immobilized metal ion chelate complex. By synthesis of a class of chelator lipids, we have transferred this approach to the concept of self-assembly. The specific interaction and lateral organization of a fluorescent fusion molecule containing a C-terminal oligohistidine sequence was studied by film balance techniques in combination with epifluorescence microscopy. Due to the phase behavior of the various lipid mixtures used, the chelator lipids can be laterally structured, generating two-dimensional arrays of histidine-tagged biomolecules. Because of the large variety of fusion proteins already available, this concept represents a powerful technique for orientation and organization of proteins at lipid interfaces with applications in biosensing, biofunctionalization of nanostructured interfaces, two-dimensional crystallization, and studies of lipid-anchored proteins.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.