The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25°C) than the eukaryotic isoenzymes after denaturation in 6 M guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.The rate of polypeptide chain elongation in prokaryotes (1, 2) is 4 -10 times faster than in eukaryotic cells (3,4). Here, we address the question of whether the faster rate of protein synthesis in prokaryotes correlates with faster rates of protein folding. The ultimate determinant of the folding rate of proteins is the primary structure including proline content. However, other factors such as protein size, chain topology, and thermodynamic stability are thought to contribute to a wide range of kinetic patterns (5-7). For our study, we chose three sets of homologous (or more precisely, orthologous) eukaryotic and prokaryotic proteins (Table I) to eliminate differences in size or chain topology. The AspATs 1 of chicken (mitochondrial and cytosolic isoenzymes) and of Escherichia coli possess nearly identical structures (8). All three enzyme variants are ␣ 2 dimers, each subunit composed of 13 ␣-helices, a 7-stranded ␤-sheet core, and one molecule of the coenzyme PLP. The two other sets of orthologous proteins that we examined were prokaryotic and eukaryotic MDH and LDH. MDH is an ␣ 2 -dimer with 8 ␣-helices and 5-6 ␤-strands/subunit, and LDH is a tetramer with 9 ␣-helices and 6 -10 ␤-strands/subunit. EXPERIMENTAL PROCEDURESProteins-Mitochondrial and cytosolic AspAT were purified from chicken heart as described previously (9). E. coli AspAT was overproduced in strain TY103 harboring pKDHE 19/AspAT (kindly provided by Dr. H. Kagamiyama, Osaka Medical College) and purified according to published protocols (10). Prior to experimentation, all AspATs were subjected to Sephadex G-25 gel filtration (Amersham Pharmacia Biotech) to remove excess cofactor PLP. The concentrations of the purified enzymes in the PLP form were determined photometrically (⑀ 280 ϭ 4.7ϫ10 4 M Ϫ1 cm Ϫ1 for E. coli AspAT and ⑀ 280 ϭ 7.0ϫ10 4 M Ϫ1 cm Ϫ1 for both mitochondrial and cytosolic AspATs; Ref. 9). Porcine mitochondrial, porcine cytosolic and E. coli MDH, as well as chicken muscle (type XXXIV), chicken heart (type VIII), and Bacillus stearothermophilus LDH, were purchased from Sigma.Denaturation and Refolding-Denaturation of AspAT was verified by determining th...

The secondary structure of bacteriorhodopsin polypeptides comprising two (AB, CD, DE, FG), three (AC, CE, EG), four (AD, DG), or five (AE, CG) of the seven transmembrane segments has been analyzed by circular dichroism spectroscopy. A comparison of the ␣-helix content with that predicted from the high resolution structure of the native protein revealed that the N-terminal AB, AC, AD, and AE fragments and the C-terminal CG fragment are completely refolded in the presence of mixed phospholipid micelles. In contrast, the DG, EG, FG, CD, CE, and DE fragments did not form ␣-helices of the expected lengths at pH 6. Each of the latter fragments displayed, however, an increased helicity upon lowering the pH to 4. Fluorescence measurements with the CD and FG fragments suggest that this helix formation occurs within transmembrane segments C and G, respectively, and thus is likely to originate from the protonation of carboxyl residues that participate in proton translocation. The partial misfolding at neutral pH observed for the shorter fragments from the central and C-terminal part of bacteriorhodopsin indicates that the conformation of some transmembrane segments is specified by interactions with neighboring helices in the assembled structure. Moreover, the data demonstrate that two stable helices at the N terminus of a multihelical membrane protein are sufficient as a folding template to induce a native conformation to the following transmembrane domains.Integral membrane proteins generally adopt a regular secondary structure within the lipid bilayer in order to satisfy hydrogen bonding of the peptide backbone in a hydrophobic environment. The physicochemical constraints imposed by the membrane limit the variety of basic protein architectures, and structure prediction is thus expected to be much simpler than for globular proteins. This characteristic has been exploited in the development of different algorithms for the identification of membrane protein secondary structure (1-3). Transmembrane (TM) 1 helices represent the predominant structural element and can be predicted with high reliability by scanning the protein sequence for regions of adequate length and hydrophobicity to span the lipid bilayer in an ␣-helical conformation. One such algorithm identifies TM ␣-helices based on an estimate of the free energy of transferring a helical segment from the aqueous environment into a lipid bilayer (3). These thermodynamic calculations predict that the individual TM ␣-helix should be stable, even in the absence of interactions with other parts of the molecule. The folding and assembly of multihelical membrane proteins could thus proceed according to a sequential two-step mechanism, in which the individual TM helices form during the initial membrane insertion step, and subsequently associate to form the native tertiary structure (4, 5). Direct structural studies on integral membrane proteins are hindered by the difficulty in obtaining crystals suitable for x-ray diffraction, and by their restricted motion in the lipid enviro...

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-l, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-l and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.

Integrative Lerntherapie ist die Hilfeform für Kinder, Jugendliche und selten auch Erwachsene, die besondere Schwierigkeiten haben, lesen, schreiben und/oder rechnen zu lernen. Der oft erhebliche Rückstand gegenüber der Altersgruppe trotz großen Lernaufwandes erschüttert mehr und mehr das Zutrauen dieser Kinder und Jugendlichen in die eigene Lernfähigkeit. Dies kann über den betroffenen Bereich hinaus bis hin zu erheblichen Beeinträchtigungen ihrer sozialen Integration und psychischen Stabilität führen. Auch außerhalb des schulischen Rahmens werden die betroffenen jungen Menschen in ihrem Alltag ständig mit ihren Erschwernissen konfrontiert, da es sich um Kulturtechniken handelt, deren Beherrschung in der Alltagsbewältigung und -gestaltung eine fundamentale Rolle spielt. Die Lerntherapie hat deshalb das Ziel, die seelische Gesundheit des Kindes oder Jugendlichen wieder herzustellen und zugleich Lernfortschritte in den betroffenen Kulturtechniken zu ermöglichen. Beratung und Begleitung des beteiligten Umfeldes, in der Regel Eltern, Lehrer und Lehrerinnen, gehören zu den Aufgabenbereichen der Lerntherapie, werden hier aber nicht im Zentrum stehen. Mit den folgenden Überlegungen wollen wir darstellen, wie in der konkreten fachbezogenen Arbeit an einem oft negativ besetzten Bereich eine Veränderung der von den leidvollen Erfahrungen geprägten Sicht unterstützt werden kann. Wir werden sprachliche Angebote darstellen, die sich an die Persönlichkeit des Kindes richten. Die Kinder sollen ihre vorhandenen Kompetenzen wahrnehmen und ihre negativen Bewertungen allmählich verändern. Mit sorgfältiger sprachlicher Begleitung gestalten wir den Lernprozess so, dass Kinder zu Entdeckern werden und durch lautes Denken ihr Wissen begleiten und neu sortieren. Neben Darlegungen und Beispielen zu unserem Vorgehen werden wir abschließend zeigen, wie Metaphern und Analogien diesen Prozess beim Kind bereichern können.

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