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To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances.
The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25C the tetramerdimer dissociation constants for each single-mutant enzyme are similar, about 10 -6 M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramermonomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10- 15 M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10-28 M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.
Die Einführung der Städteordnung 1808 wird den preußischen Reformern unter Stein und Hardenberg als wichtiger Schritt zur städtischen Selbstverwaltung zugerechnet. Die Bedeutung der Städteordnung ist in der Wissenschaft und Praxis unbestritten. Sie gilt als die erste Konstituierung des modernen Gemeindeverfassungsrechts. Mit der Schaffung der Stadtverordnetenversammlung und der damit verbundenen Einführung des freien Mandats der Mitglieder trug sie die Merkmale des ersten modernen „Parlaments“ in Deutschland. Die Stein’sche Städteordnung gewährte der Stadtverordnetenversammlung ein hohes Maß an Autonomie, insbesondere Budget- und Steuerbewilligungsrecht, ohne an staatliche Gesetzesnormen gebunden zu sein. Auch in Potsdam wurde im Zuge der preußischen Reformen im März 1809 erstmals eine Stadtverordnetenversammlung gewählt. Das 200jährige Jubiläum war für Wissenschaftler und Experten aus Politik und Verwaltung Anlass, verfassungsrechtliche Grundlagen, Entscheidungskompetenzen, Aufgaben und Strategien der Stadtverordnetenversammlung im Wandel der Zeit zu diskutieren.
We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 ;s. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.