Directed evolution study of temperature adaptation in a psychrophilic enzyme.

We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins.

Thermodynamic stability of archaeal histones.

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is > 90% reversible, with concentration-dependent apparent Tm values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyA1 dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing < 200 mM, < 400 mM, and < 1.

Archaeal nucleosomes.

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers.

DNA binding and nuclease protection by the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus.

The DNA-binding and nuclease-protection properties of the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus have been shown to be consistent with the formation of nucleosome-like structures (NLS). These proteins bind to DNA molecules as short as 20 bp and form complexes that protect DNA fragments from micrococcal nuclease (MNase) digestion that are 30 bp, approximately 60 bp and multiples of approximately 60 bp in length. The sequences of 49 of the approximately 60-bp DNA fragments protected from MNase digestion by HMfA have been determined and their intrinsic curvatures calculated.

Histones and chromatin structure in hyperthermophilic Archaea.

HMf is a histone from the hyperthermophile Methanothermus fervidus. It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome-like structures (NLS). HMf preparations are mixtures of two similar, small (approximately 7.