Transportprozesse und molekulare Wechselwirkungen : Untersuchungen biologischer Systeme mittels Einzelmolekül-Fluoreszenzmethoden

Durch die Entschlüsselung des menschlichen Genoms wurde eine Fülle detaillierter Bauanleitungen von Proteinen - den Grundbausteinen des Lebens - bekannt. Um dynamische Prozesse, wie Transportvorgänge und molekulare Wechselwirkungen, in lebenden Zellen zu untersuchen, muss aber auf andere Techniken als die der klassischen Biochemie zurückgegriffen werden. In jüngerer Zeit haben sich hierbei vor allem Methoden der Einzelmolekülfluoreszenzspektroskopie als wegweisend herausgestellt. Ein wichtiger Transportmechanismus in Zellen ist die Diffusion, die durch Größe, Form, Ladung und durch die hohe Konzentration der beteiligten Proteine beeinflusst wird. Zum besseren Verständnis dieser Einflüsse wurde in dieser Arbeit die Diffusion von Proteinen bei physiologischen Proteinkonzentrationen mit Hilfe der Fluoreszenz-Korrelations-Spektroskopie untersucht. Der Vergleich der gemessenen Diffusionskoeffizienten mit Simulationen legt nahe, dass spezifische Proteinstrukturen für abweichendes Verhalten unterschiedlicher Proteinlösungen ausschlaggebend sind. Diese Arbeit liefert Ansätze für eine systematische Studie der beteiligten Parameter zum besseren Verständnis der molekularen Wechselwirkungen in lebenden Zellen. Neben der Diffusion von Proteinen im Zytoplasma wurde auch die Mobilität von Membranproteinen, die für die zelluläre Reaktion auf äußere Einflüsse wichtig sind, in Pflanzenzellen untersucht. Die gemessenen Diffusionskoeffizienten des Kalium-Kanals KAT1 können durch die Existenz von Mikrodomänen erklärt werden. Die Beweglichkeit in der Plasmamembran lässt sich mit Lipiden vergleichen. Außerdem wurde eine neue Methode zur Charakterisierung der Proteinhülle von Transportvesikeln entwickelt, die, im Gegensatz zum stochastischen Markierungsansatz über Immunogoldpartikel, die auftretenden Isoformen spektral unterscheiden kann. Zur Analyse der Zusammensetzung wurde eine Kolokalisationsanalyse mit alternierender Laseranregung kombiniert. Dieser Methodenansatz liefert damit einen neuen Anstoß zur quantitativen Untersuchung der Proteinbeladung von Vesikeln

The Shape of Protein Crowders is a Major Determinant of Protein Diffusion

AbstractAs a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 μs), appears only at very short timescales (<1 μs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions

Genome-wide analysis identifies 12 loci influencing human reproductive behavior.

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits

Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis

The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (K(D)) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse

Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis

The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (K(D)) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse