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.
D-Glyceraldehyde3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH) is intrinsically thermostable, exhibiting a thermal transition beyond 105°C. Neither the amino-acid composition nor homology modeling based on sequence alignment and known three-dimensional structures of the enzyme from meso- and thermophiles provide an explanation of the anomalous stability. Recent X-ray data (Korndörfer et al., 1994) suggest an increased number of ion-pairs to be involved. To prove this hypothesis, a number of charged residues contributing to ion-pairs in TmGAPDH, but absent in the moderately thermophilic enzyme were altered. Elimination of peripheral ion-pairs (E103-K104, E261-R266) was found to be ineffective. Altering a central charge cluster (R10-D47, E314, D*186) lead to a drastic decrease in coenzyme binding. As a consequence, guanidine-dependent deactivation is shifted to significantly lower GdmCl concentrations without altering the denaturation/dissociation profile of the wildtype enzyme. Mutants in the S-loop (R195D, R195D/D181K) lead to a biphasic profile in the GdmCl-dependent denaturation transition and significant destabilization; at room temperature no subunit dissociation can be observed.
The past years have seen immense efforts in the structure prediction area, with a strong bias for the development of algorithms and computer programs that are used for practical applications (protein design, structure analysis). Knowledge-based modeling techniques are still the best choice for structure prediction if reliable and reasonable model structures are needed within short time, and these methods continue to be investigated and refined. Also, there is a strong and increasing demand for world-wide, high-speed networking for information access i. e. via the Internet. Publicly available computer programs, free of charges for the academic community, may speed up the spreading of new ideas and methodologies, and may also be useful for the assessment of the general applicability of these new approaches. However, there is still no unambiguous and faultless method for the prediction of the structure, function, and other properties of a protein or other biological macromolecule from its building blocks. The protein folding problem will thus continue to be an exciting, but often also discouraging, area of research.
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Dihydrofolate reductase (Dhfr) from the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. Sequence determination of the reported dyrA gene was repeated, and a corrected version deposited in the nucleotide databank (accession number Y11021). Ultracentrifugational analysis and gel permeation chromatography prove the enzyme to form a stable homo‑dimer. The enzyme exhibits long‑term stability at physiological temperature (80°C), and in the presence of high denaturant concentrations (half‑time in 6 M guanidinium chloride: 1d). Alignments of Dhfrs from different species, as well as comparative modeling based on the homology to the crystal structures of the enzyme from prokaryotes and eukaryotes, were used to generate a model of the three‑dimensional structure. The apoenzyme was crystallized and a data set was collected to a resolution of about 2 Å.