DNA G-quartets in a 1.86 A resolution structure of an Oxytricha nova telomeric protein-DNA complex.

The Oxytricha nova telomere end binding protein (OnTEBP) recognizes, binds and protects the single-stranded 3'-terminal DNA extension found at the ends of macronuclear chromosomes. The structure of this complex shows that the single strand GGGGTTTTGGGG DNA binds in a deep cleft between the two protein subunits of OnTEBP, adopting a non-helical and irregular conformation. In extending the resolution limit of this structure to 1.86 A, we were surprised to find a G-quartet linked dimer of the GGGGTTTTGGGG DNA also packing within the crystal lattice and interacting with the telomere end binding protein. The G-quartet DNA exhibits the same structure and topology as previously observed in solution by NMR with diagonally crossing d(TTTT) loops at either end of the four-stranded helix. Additionally, the crystal structure reveals clearly visible Na(+), and specific patterns of bound water molecules in the four non-equivalent grooves. Although the G-quartet:protein contact surfaces are modest and might simply represent crystal packing interactions, it is interesting to speculate that the two types of telomeric DNA-protein interactions observed here might both be important in telomere biology.

The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA.

Telomeres are specialized protein-DNA complexes that compose the ends of eukaryotic chromosomes. Telomeres protect chromosome termini from degradation and recombination and act together with telomerase to ensure complete genome replication. We have determined the crystal structure of the two-subunit Oxytricha nova telomere end binding protein (OnTEBP) complexed with single strand telomeric DNA at 2.8 A resolution. The structure reveals four oligonucleotide/oligosaccharide-binding folds, three of which form a deep cleft that binds the ssDNA, and a fourth that forms an unusual protein-protein interaction between the alpha and beta subunits. This structure provides a molecular description of how the two subunits of OnTEBP recognize and bind ssDNA to form a sequence-specific, telomeric nucleoprotein complex that caps the very 3' ends of chromosomes.

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo characterization of mutants of the bacteriophage f1 gene V protein isolated by saturation mutagenesis.

The gene V protein of bacteriophage f1 binds to single-stranded nucleic acids and is essential for propagation of phage f1. We tested the function of gene V protein mutants with single amino acid substitutions in two ways: by the ability of the mutant proteins to support phage growth, and by the ability of the mutant proteins, when expressed at high levels, to inhibit the growth of Escherichia coli. The results of the tests were used to identify sites in the protein that are relatively tolerant or intolerant to substitution, where tolerant sites are defined as those where most substitutions do not affect the function of the protein. The two assays generally yielded similar results for the tolerance of sites to substitution. Many sites that are less than 10% exposed to the solvent are relatively intolerant of substitution, with even very conservative substitutions leading to loss of function in some cases such as Ile to Leu at residue 6. Some buried sites such as Ile47 are more tolerant, with even a substitution of Ile to Thr leading to a functional protein based on the ability of the proteins to inhibit the growth of E. coli. Some surface sites in the protein (> 10% exposure to solvent) that are thought to be near the location of bound oligonucleotides, such as Arg16, Val19, Ser20, Arg21, Tyr26, Lys46 and Arg80, are sensitive to substitution. Other side-chains thought to be close to bound oligonucleotides, including Leu28, can be replaced with a number of amino acids with little loss of function based on either assay. Most non-Gly/Pro surface residues thought to be distant from the locations of bound oligonucleotides are relatively tolerant of substitution, except for two small residues (Ala11 and Thr14), two aromatic residues (Tyr34 and Tyr56), two residues that are only partially exposed to solvent (Asn29 and Val70), and three residues that have been proposed to be at the dimer-dimer interface formed when gene V protein binds to nucleic acids (Glu40, Tyr41 and Arg82).

Approaches to predicting effects of single amino acid substitutions on the function of a protein.

The relative activities of 313 mutants of the gene V protein of bacteriophage f1, assayed in vivo, have been used to evaluate two approaches to predicting the effects of single amino acid substitutions on the function of a protein. First, we tested methods that only depend on the properties of the wild-type and substituting amino acids. None of the properties or measures of the functional equivalence of amino acids we tested, including the frequency of exchange of amino acids among homologous proteins as well as changes in side-chain size, hydrophobicity, and charge, were found to be more than weakly correlated with the activities of mutants. The principal reason for this poor correlation was found to be that the effect of a particular substitution varies considerably from site to site. We then tested an approach using the activities of several mutants with substitutions at a site to predict the activity of another mutant, and we find that this is a relatively good indicator of whether the other mutant at that site will be functional. A predictive scheme was developed that combines the weak information from the models depending on the properties of the wild-type and substituting amino acids with the stronger information from the tolerance of a site to substitution. Although this scheme requires no knowledge of the structure of a mutant protein, it is useful in predicting the activities of mutants.