Cold shock proteins (Csp) are small acidic proteins that fold into ??-barrel structures with five anti-parallel ??-strands and are involved in essential cellular processes. Upon temperature downshift the synthesis of Csp proteins is drastically increased to enable cells to restore growth in the cold. These proteins facilitate transcription and translation at low temperature by functioning as RNA chaperones. Csp proteins have been most extensively studied in Bacteria but very few Csp homologues have been identified and studied in Archaea. This is the first study examining structural, functional and biophysical properties of Csp from the Antarctic archaeon Methanogenium frigidum. The fastidious growth requirements of M. frigidum make it difficult to cultivate, therefore recombinant methods have been developed for the expression and characterization of the protein. The analysis by transverse urea gradient gel electrophoresis (TUG-GE) revealed that M. frigidum Csp folds by a reversible two-state mechanism and has a low conformational stability. The spectroscopic analysis of the protein performed by Circular Dichroism (CD) spectroscopy disclosed features typical of other homologous proteins. A possible association between Csp and RNA has been proposed according to MALDI-TOF mass spectrometry analysis. The effect of a Nterminal polyhistidine affinity tag on the biophysical properties of Csp was also examined. The biological activity of Csp was investigated by complementation of an E. coli cold sensitive mutant. These studies revealed that the M. frigidum Csp is biologically active and can function in E. coli.

Cold shock proteins (Csp) are small acidic proteins that fold into ??-barrel structures with five anti-parallel ??-strands and are involved in essential cellular processes. Upon temperature downshift the synthesis of Csp proteins is drastically increased to enable cells to restore growth in the cold. These proteins facilitate transcription and translation at low temperature by functioning as RNA chaperones. Csp proteins have been most extensively studied in Bacteria but very few Csp homologues have been identified and studied in Archaea. This is the first study examining structural, functional and biophysical properties of Csp from the Antarctic archaeon Methanogenium frigidum. The fastidious growth requirements of M. frigidum make it difficult to cultivate, therefore recombinant methods have been developed for the expression and characterization of the protein. The analysis by transverse urea gradient gel electrophoresis (TUG-GE) revealed that M. frigidum Csp folds by a reversible two-state mechanism and has a low conformational stability. The spectroscopic analysis of the protein performed by Circular Dichroism (CD) spectroscopy disclosed features typical of other homologous proteins. A possible association between Csp and RNA has been proposed according to MALDI-TOF mass spectrometry analysis. The effect of a Nterminal polyhistidine affinity tag on the biophysical properties of Csp was also examined. The biological activity of Csp was investigated by complementation of an E. coli cold sensitive mutant. These studies revealed that the M. frigidum Csp is biologically active and can function in E. coli.

Thesis (doctoral)--Universität Potsdam, 2007. / Includes bibliographical references (p. 71-82).

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed Nov. 9, 2007). PDF text: xii, 144 p. : ill. ; 9 Mb. UMI publication number: AAT 3263486. Includes bibliographical references. Also available in microfilm and microfiche formats.

Univ., Diss.--Potsdam, 2007. / Enth. außerdem 4 Papers.

No description available.

The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo / universal&rdquo / archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo / universal&rdquo / archaeal primers A571F (5&rsquo / -GCY TAA AGS RIC CGT AGC-3&rsquo / ) and UA1204R (5&rsquo / -TTM GGG GCA TRC IKA CCT-3&rsquo / ) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.

The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo / universal&rdquo / archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo / universal&rdquo / archaeal primers A571F (5&rsquo / -GCY TAA AGS RIC CGT AGC-3&rsquo / ) and UA1204R (5&rsquo / -TTM GGG GCA TRC IKA CCT-3&rsquo / ) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

PIB-ATPases are membrane proteins that transport heavy metal ions across biological membranes upon ATP-hydrolysis. These enzymes contribute to metal homeostasis in archaeal, prokaryotic and eukaryotic cells. Typically, most PIB-ATPases have eight transmembrane segments, one or more metal binding domains in the cytoplasmic N-terminal region and a series of amino acids conserved in all the members of this family. By sequence homology analysis, the metal specificity for most ATPases has been predicted. Here, we report studies on PIB-ATPases from different organisms. The first part of this work focuses in a group of ATPases from Arabidopsis thaliana plants. Transcription levels of HMA3, 4 and 8 were analyzed in different plant organs and in seedlings upon metal exposure. Tissue specificity was studied for HMA8 by generation of transgenic plants carrying a reporter gene downstream its promoter region. Attempts to determine metal specificity of proteins expressed in yeast cells were performed. Finally, in order to study the effects of removing the genes products from the plants, HMA4 and 8 mutant plants were identified. The second part describes a novel Pb-transport ATPase from a thermophilic archaeabacterium, Aeropyrum pernix. This enzyme is predicted to have only six transmembrane segments, no regulatory metal binding domains and unusual metal specificity. PbTP was cloned, expressed in Escherichia coli and partially purified. The enzyme retained its thermophilicity characteristics when isolated from its native lipid environment. The metal dependent ATPase activity was determined in the presence of different metals at 75ºC. The enzyme was highly activated by Pb2+ (Vmax: 23.6 µmol Pi/mg/h) and to a lesser extent by Zn2+, Hg2+ and Cd2+. Lead interacts with PbTP with high apparent affinity (K1/2: 4.6 µM). The enzymatic ATP hydrolysis was independent of cysteine or glutathione, suggesting direct interaction of the metal ions with the transmembrane transport sites.