An accessible hydrophobic surface is a key element of the molecular chaperone action of Atp11p.

Atp11p is a soluble protein of mitochondria that binds unassembled beta subunits of the F(1)-ATPase and prevents them from aggregating in the matrix. In this report, we show that Atp11p protects the insulin B chain from aggregating in vitro and therefore acts as a molecular chaperone. The chaperone action of Atp11p is mediated by hydrophobic interactions. An accessible hydrophobic surface in Atp11p was identified with the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5-napththalenesulfonic acid (bis-ANS). The spectral changes of bis-ANS in the presence of Atp11p indicate that the probe binds to a nonpolar region of the protein. Furthermore, the dye quenches the fluorescence of Atp11p tryptophan residues in a concentration-dependent manner. Although up to three molecules of bis-ANS can bind cooperatively to Atp11p, the binding of only one dye molecule is sufficient to virtually eliminate the chaperone activity of the protein.

The alpha-subunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p.

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F(1) component of the mitochondrial F(1)F(0) ATP synthase. In this report, we show that the F(1) alpha-subunit co-precipitates and co-purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F(1) alpha-subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the alpha-subunit. Yeast two-hybrid screens confirm the direct association of Atp12p with the alpha-subunit and indicate that the binding site for the assembly factor lies in the nucleotide-binding domain of the alpha-subunit, between Asp133 and Leu322. These studies provide the basis for a model of F(1) assembly in which Atp12p is released from the alpha-subunit in exchange for a beta-subunit to form the interface that contains the non-catalytic adenine nucleotide-binding site.

Lack of correlation between binding of EcoRII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations.

The cytosine methyltransferases (MTases) M. HhaI and M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent Kd for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells.

Cytosine methyltransferase from Escherichia coli in which active site cysteine is replaced with serine is partially active.

EcoRII methyltransferase (M.EcoRII) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to C-5 position of second cytosine in the DNA sequence 5'-CCWGG (W = A or T). The reaction is initiated by a nucleophilic attack of the C-6 of target cytosine by a cysteine that is conserved among all cytosine methyltransferases. We have replaced this cysteine in M.EcoRII with serine or alanine and purified the proteins to homogeneity. The catalytic efficiency (kcat/Km) of the mutant enzyme with serine (C186S) for methyl transfer is about 10,000 times less than that of WT but is substantially higher than the efficiency of the C186A mutant. We show that the WT enzyme and C186S mutant are proficient in exchange of proton at C-5 and that this activity is reduced in the mutant to the same extent as the methyl transfer activity. The C186S mutant is insensitive to a cysteine-specific inhibitor, and it transfers methyl groups to the same position of cytosine as the WT enzyme. The ability of serine to act as a nucleophile in the enzyme reaction suggests that it--and probably the cysteine in the WT enzyme--is activated by a nearby base. Like the WT enzyme, C186S forms stable SDS-resistant complexes with DNA containing 5-azacytosine; but unlike the WT enzyme, the mutant reacts faster with 5-azacytosine than with normal cytosine. Apparently, greater reactivity of 5-azacytosine assists the C186S mutant in catalysis.

Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements.

Two mutants of bacterial formate dehydrogenase from Pseudomonas sp.101 (EC 1.2.1.2, FDH)-C255S (FDH-S) and C255M (FDH-M), were obtained and its properties were studied. Both mutations provided the high resistance to inactivation by Hg2+. Slow inactivation of mutants by DTNB reveals the presence in FDH molecule of another essential cysteine residue. Specific activities of FDH, FDH-S and FDH-M were 16, 16 and 9.5 U/mg of protein, respectively. Km on formate was 7.5, 7.5 and 20 mM and Km on NAD(+)-0.1, 0.3 and 0.6 mM for FDH, FDH-S and FDH-M, respectively. Mutations of Cys255 on Ser or Met resulted in increasing of enzyme stability at 25 degrees C and decreasing of thermostability (above 45 degrees C). Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.