Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti: a combined molecular biology and force spectroscopy investigation

Bartels FW, Baumgarth B, Anselmetti D, Ros R, Becker A. Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti: a combined molecular biology and force spectroscopy investigation. Journal of structural biology. 2003;143(2):145-152.Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28 bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000 pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of k off = (1.2 ± 1.0) × 10 -3 s -1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism

Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Baumgarth B, Bartels FW, Anselmetti D, Becker A, Ros R. Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA. Microbiology. 2005;151(1):259-268.The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction

Force spectroscopy of single protein-DNA complexes

Bartels FW. Kraftspektroskopische Bindungsstudien an einzelnen Protein-DNA-Komplexen. Bielefeld (Germany): Bielefeld University; 2005.Specific protein-DNA interaction is fundamental for all aspects of gene expression. A regulatory model system is the biosynthesis of exopolysaccharides (EPS) in the nitrogen-fixating bacterium Sinorhizobium meliloti 2011. These sugar polymers promote the bacterium's symbiosis with alfalfa plants, a process of agricultural importance. The EPS biosynthesis is controlled by a complex interplay of several proteins, most prominently the transcriptional activator ExpG. Using single molecule experiments, this work demonstrates that the protein ExpG binds to three different DNA target sequences in a sequence specific manner, albeit with distinct differences in the energy landscape. Dynamic force spectroscopy based on the atomic force microscope (AFM) proves to be sensitive even to small variations of the binding motif. Experiments with DNA mutants lead to a deeper understanding of the binding mechanism. The method is then applied to a more complex interaction from the same regulatory system. The binding of the quorum sensing receptor ExpR to its DNA target sequence is stimulated by an acyl-homoserine lactone (AHL) effector molecule. Dynamic force spectroscopy yields detailed information about the AHL spectrum, revealing differences in the energy landscape of the protein-DNA interaction depending on the stimulating effector.Spezifische Protein-DNA-Interaktion ist von grundlegender Bedeutung für alle Aspekte der Genexpression. Ein regulatorisches Modellsystem stellt die Biosynthese von Exopolysacchariden (EPS) im Bakterium Sinorhizobium meliloti 2011 dar. Diese Zuckerpolymere befördern die Symbiose des Bakteriums mit Alfalfa-Pflanzen, die der Fixierung von molekularem Stickstoff dient - ein Prozess von landwirtschaftlicher Bedeutung. Die EPS-Biosynthese wird von einem komplexen Wechselspiel mehrerer Proteine kontrolliert, unter denen der Transkriptionsaktivator ExpG hervorsticht. Durch Einzelmolekülexperimente weist diese Arbeit nach, dass das Protein ExpG sequenzspezifisch an drei verschiedene DNA-Zielsequenzen bindet. Dabei zeigen sich jedoch charakteristische Unterschiede in der Energielandschaft der Interaktion. Dynamische Kraftspektroskopie basierend auf dem Rasterkraftmikroskop (Atomic Force Microscope, AFM) ist in der Lage, selbst kleine Variationen des Bindemotivs zu detektieren. Experimente mit DNA-Mutanten führen dann zu einem tieferen Verständnis in Bezug auf den Mechanismus der Bindung. Die Methode wird anschließend auf eine komplexere Wechselwirkung aus demselben regulatorischen System angewendet. Die Bindung des Quorum-Sensing-Rezeptors ExpR an seine DNA-Zielsequenz wird durch Acyl-Homoserinlacton(AHL)-Effektoren stimuliert. Dynamische Kraftspektroskopie liefert Detailinformationen über das AHL-Spektrum: Es zeigen sich Unterschiede in der Energielandschaft der Protein-DNA-Interaktion in Abhängigkeit des stimulierenden Effektors

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy

Anselmetti D, Bartels FW, Becker A, et al. Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy. Langmuir. 2008;24(4):1365-1370.Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future

Effector-Stimulated Single Molecule Protein-DNA Interactions of a Quorum-Sensing System in Sinorhizobium meliloti

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 Å. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction