ABSTRACT
GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.
Subject(s)
Chloroplasts/chemistry , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Membrane Proteins/chemistry , Pisum sativum/chemistry , Plant Proteins/chemistry , Protein Precursors/chemistry , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Plant Proteins/genetics , Protein Binding , Protein Multimerization , Protein Precursors/genetics , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Substrate Specificity , ThermodynamicsABSTRACT
Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures.
Subject(s)
Carbocyanines/chemistry , DNA/chemistry , Spectrometry, Fluorescence , Bayes Theorem , Inverted Repeat Sequences , KineticsABSTRACT
Systematic studies are presented demonstrating the complementarity of directed ortho metalation (DoM) and Ir-catalyzed strategies for the provision of borylated aromatics and their subsequent Suzuki-Miyaura coupling reactions. A new concept, the use of the TMS group, readily introduced by DoM, as a latent regiodirective moiety to overcome the otherwise problematic production of isomeric borylated product mixtures is presented. Additional electrophile-induced ipso-deborylation and DoM reactions of the Bpin products are described.
Subject(s)
Boron Compounds/chemistry , Heterocyclic Compounds/chemistry , Hydrocarbons, Aromatic/chemistry , Iridium/chemistry , Metals/chemistry , Catalysis , Hydrogen Bonding , Molecular Structure , StereoisomerismABSTRACT
Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid-a process that is driven by one of the most powerful molecular motors.
