Hier finden Sie alle wissenschaftlichen Publikationen und Arbeiten bzw. Veröffentlichungen von Dr. Gerald Böhm aus der Zeit von 1988 bis 2007. Die PDF-Dateien stehen zum Download gemäß den jeweiligen Urheberrechten bereit.

Three complete genome sequences of thermophilic bacteria provide a wealth of information challenging current ideas concerning phylogeny and evolution, as well as the determinants of protein stability. Considering known protein structures from extremophiles, it becomes clear that no general conclusions can be drawn regarding adaptive mechanisms to extremes of physical conditions. Proteins are individuals that accumulate increments of stabilization; in thermophiles these come from charge clusters, networks of hydrogen bonds, optimization of packing and hydrophobic interactions, each in its own way. Recent examples indicate ways for the rational design of ultrastable proteins.

For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red). Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E. coli. Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild type-protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.

The histone-like protein TmHU from the hyperthermophilic eubacterium Thermotoga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromatography. CD spectroscopical studies with secondary structure analysis as well as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The Tm of the protein was determined to be 96 °C, and thermal unfolding is nearly completely reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a KD of 73 nM and a Hill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated that TmHU is capable to increase the melting point of a synthetic, double-stranded DNA (poly[d(A-T)]) by 47 °C, thus suggesting that DNA stabilization may be a major function of this protein in hyperthermophiles. The significant in vitro protection of double-helical DNA may be useful for biotechnological applications.

Thermotoga maritima (Tm) expresses a 7 kDa monomeric protein whose 18 N-terminal amino acids show 81% identity to N-terminal sequences of cold shock proteins (Csps) from Bacillus caldolyticus and Bacillus stearothermophilus. There were only trace amounts of the protein in Thermotoga cells grown at 80°C. Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli. A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N-terminus of TmCsp and the known C-terminus of CspB from Bacillus subtilis. Southern blot analysis of genomic Tm DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene-and vector-specific primers to identify the complete DNA sequence. As reported for other csp genes, the 5′ untranslated region of the mRNA was anomalously long; it contained the putative Shine–Dalgarno sequence. The coding part of the gene contained 198 bp, i.e., 66 amino acids. The sequence showed 61% identity to CspB from B. caldolyticus and high similarity to all other known Csps. Computer-based homology modeling allowed the conclusion that TmCsp represents a β-barrel similar to CspB from B. subtilis and CspA from E. coli.

As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, over-expression of the recombinant protein yielded authentic TmCsp with a molecular weight of 7,474 Da. This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein. The temperature-induced equilibrium transition at 87 °C exceeds the maximum growth temperature of Tm and represents the maximal Tm-value reported for Csps so far.

VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro. Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid dissembly.

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