Ligand-Specific Interactions Modulate Kinetic, Energetic, and Mechanical Properties of the Human β2 Adrenergic Receptor

SummaryG protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β2 adrenergic receptor (β2AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β2AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns

Structure of the Extracellular Portion of CD46 Provides Insights into Its Interactions with Complement Proteins and Pathogens

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system

Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design

Novel single-molecule force spectroscopy approaches to characterize interactions of membrane proteins

Abstract: Atomic force microscopy (AFM) based single-molecule force spectroscopy (SMFS) is a biophysical tool used to investigate folding and unfolding of biological macromolecules, like membrane proteins. Unfolding of single membrane proteins can be recorded by force-distance (FD) curves, which exhibit reproducible sawtooth-like patterns of force peaks. These force peaks reflect the unfolding of stable structural segments. In the case of α-helical transmembrane proteins, these segments consist of partial or complete α-helices, or even of several consecutive α-helices connected by extracellular or intracellular loops. Fitting these force peaks using polymer extension models reveals the exact position of the interaction within the membrane protein. Furthermore, with SMFS based dynamic force spectroscopy (DFS) it is possible to study intrinsic behavior of proteins, such as energetic, kinetic and mechanical properties, or, in other words, their energy landscape. The work presented here contains two SMFS-related projects that were carried out independently from each other. However, both projects are novel SMFS approaches that improve our understanding of α-helical transmembrane proteins. In the first project, it was investigated how cholesterol, an essential component of eukaryotic membranes, and ligands modulate the energy landscape of the human β2 adrenergic G protein-coupled receptor (β2AR). G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Environmental changes induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of β2AR was addressed in this project. Cholesterol considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and function relationship of β2AR. One exception was the structural core segment of β2AR, which establishes multiple ligand-binding sites and which properties were not significantly influenced by cholesterol. This suggests that cholesterol may not necessarily influence ligand binding to β2AR rather than setting the GPCR into a different state so that the receptor will respond differently to ligand binding. For that purpose, SMFS and DFS approaches were used to investigate how ligand binding modulates the energy landscape of β2AR. Five different ligands that represented agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic and mechanical properties of functionally important structural regions of β2AR. These interactions were specific to the efficacy profile of the investigated ligands, which suggests that the functional modulation of GPCRs follows structurally well-defined interaction patterns. The second project addressed the problem that SMFS is a rather time-consuming technique, since the membranes embedding the membrane proteins must be imaged and localized before starting the actual SFMS measurement. In order to simplify the investigation of membrane proteins by SMFS the light-driven proton pump bacteriorhodopsin (BR) was reconstituted into lipid nanodiscs. The advantage of using nanodiscs is that membrane proteins can be handled and characterized like water-soluble proteins with similar ease. SMFS characterization of BR in native purple membranes and in nanodiscs revealed no significant alterations of structure, function, unfolding intermediates, and strengths of inter- and intra-molecular interactions. This demonstrates that lipid nanodiscs provide a unique approach for in vitro studies of native membrane proteins using SMFS and opens up a new avenue to characterize membrane proteins by a wide variety of SMFS approaches that have been established on water-soluble proteins. ---------- Zusammenfassung: Rasterkraftmikroskopie (AFM) basierte Einzelmolekül-Kraftspektroskopie (SMFS) ist eine biophysikalische Anwendung, die es ermöglicht, Entfaltung und Faltung von biologischen Makromolekülen, zum Beispiel von Membranproteinen, zu studieren. Die Entfaltung von einzelnen Makromolekülen kann mittels einer Kraft-Abstands-Kurve gemessen werden. Eine typische Kraft-Abstands-Kurve, welche die Entfaltung eines Transmembranproteins widerspiegelt, weist eine sägezahnartige Struktur aus Peaks auf. Jeder dieser Peaks entspricht der Entfaltung eines stabilen strukturellen Segments des entfalteten Proteins. Bei α-helikalen Transmembranproteinen bestehen diese Segmente aus α-Helices (oder Teilen davon), oder sogar aus mehreren Transmembransegmenten, welche durch extra- oder intrazelluläre Loops miteinander verbunden sind. Die Peaks können mittels physikalischer Modelle, die das Verhalten steifer Polymere bei Dehnung beschreiben, gefittet werden. Dadurch kann die exakte Position, an welcher innerhalb des Membranproteins eine Interaktion auftritt, bestimmt werden. Des Weiteren ist es möglich, mit dynamischer Kraftspektroskopie (DFS), ein auf SMFS basierendes Verfahren, das intrinsische Verhalten von Proteinen zu untersuchen. Beispielsweise können mittels DFS biophysikalische Parameter, wie energetische, kinetische und mechanische Eigenschaften (Energielandschaft) von Proteinen bestimmt werden. Bei der im Folgenden vorgestellten Arbeit handelt es sich um zwei voneinander unabhängig durchgeführte SMFS-Projekte. Beide Projekte sind neuartige Ansätze, welche unser Verständnis von α-helikalen Transmembranproteinen verbessern. Im ersten Projekt wurde der Einfluss von Cholesterin, einem essentiellen Bestandteil eukaryotischer Membranen, auf die Energielandschaft des humanen β2 adrenergen G-Protein-gekoppelten Rezeptors (β2AR) untersucht. G-Protein-gekoppelte Rezeptoren (GPCRs) sind die größte und vielseitigste Gruppe von Membranrezeptoren. Extrazelluläre Veränderungen induzieren inter- und intramolekulare Interaktionen, die den funktionellen Zustand von GPCRs modulieren und dadurch eine intrazelluläre Signalkaskade auslösen. In dem Projekt wurde untersucht, auf welche Art und Weise diese Interaktionen etabliert werden und wie sie den funktionellen Zustand des β2ARs beeinflussen. Cholesterin hatte einen wesentlichen Einfluss auf die Stärke der Interaktionen sowie die Energielandschaft fast aller struktureller Segmente des Rezeptors. Eine Ausnahme war das strukturelle Kernsegment von β2AR, welches eine Vielzahl von Ligandenbindungsstellen aufweist. Die Eigenschaften dieses Segmentes blieben auch in Gegenwart von Cholesterin unverändert. Da Cholesterin nicht notwendigerweise die Bindung von Liganden beeinflusst, ist zu vermuten, dass das Kernsegment seine Eigenschaften ändert, nachdem ein Ligand gebunden hat. Um diese Frage zu beantworten wurde mittels SMFS und DFS untersucht, wie die Bindung von Liganden an β2AR dessen Energielandschaft beeinflusst. Fünf Liganden unterschiedlicher therapeutischer Wirksamkeit etablierten ein Netzwerk von Interaktionen, welches die kinetischen, energetischen und mechanischen Parameter funktionell wichtiger struktureller Regionen des Rezeptors modulierte. Diese Interaktionen waren spezifisch entsprechend der Wirksamkeit des jeweiligen Liganden. Offenbar folgt die funktionelle Modulierung von GPCRs strukturell definierten Interaktionsmustern. Bei SMFS von Membranprotein handelt es sich um relativ zeitintensive Messungen, da die Membranen, in die das zu untersuchende Protein eingebettet ist, zunächst abgebildet und lokalisiert werden müssen. Dieses Problem wurde im zweiten Projekt näher betrachtet. Um SMFS mit Membranproteinen zu vereinfachen, wurde die lichtgetriebene Protonenpumpe Bakteriorhodopsin in Nanodiscs rekonstituiert. Nanodiscs sind synthetische Modellmembranen, mittels derer Membranproteine ähnlich wie wasserlösliche Proteine behandelt werden können. Die Charakterisierung von nativem BR in der Purpurmembran sowie in Nanodiscs ergab keine signifikanten Unterschiede bezüglich Struktur, Funktion, Entfaltungsintermediaten sowie Stärke von inter- und intramolekularen Interaktionen. Diese Resultate bestätigen, dass Nanodiscs neue Möglichkeiten für SMFS-Studien an Membranproteinen in vitro bieten

Luminescence from Droplet-Etched GaAs Quantum Dots at and Close to Room Temperature

Epitaxially grown quantum dots (QDs) are established as quantum emitters for quantum information technology, but their operation under ambient conditions remains a challenge. Therefore, we study photoluminescence (PL) emission at and close to room temperature from self-assembled strain-free GaAs quantum dots (QDs) in refilled AlGaAs nanoholes on (001)GaAs substrate. Two major obstacles for room temperature operation are observed. The first is a strong radiative background from the GaAs substrate and the second a significant loss of intensity by more than four orders of magnitude between liquid helium and room temperature. We discuss results obtained on three different sample designs and two excitation wavelengths. The PL measurements are performed at room temperature and at T = 200 K, which is obtained using an inexpensive thermoelectric cooler. An optimized sample with an AlGaAs barrier layer thicker than the penetration depth of the exciting green laser light (532 nm) demonstrates clear QD peaks already at room temperature. Samples with thin AlGaAs layers show room temperature emission from the QDs when a blue laser (405 nm) with a reduced optical penetration depth is used for excitation. A model and a fit to the experimental behavior identify dissociation of excitons in the barrier below T = 100 K and thermal escape of excitons from QDs above T = 160 K as the central processes causing PL-intensity loss

Single-Molecule Force Spectroscopy from Nanodiscs: An Assay to Quantify Folding, Stability, and Interactions of Native Membrane Proteins

Single-molecule force spectroscopy (SMFS) can quantify and localize inter- and intramolecular interactions that determine the folding, stability, and functional state of membrane proteins. To conduct SMFS the membranes embedding the membrane proteins must be imaged and localized in a rather time-consuming manner. Toward simplifying the investigation of membrane proteins by SMFS, we reconstituted the light-driven proton pump bacteriorhodopsin into lipid nanodiscs. The advantage of using nanodiscs is that membrane proteins can be handled like water-soluble proteins and characterized with similar ease. SMFS characterization of bacteriorhodopsin in native purple membranes and in nanodiscs reveals no significant alterations of structure, function, unfolding intermediates, and strengths of inter- and intramolecular interactions. This demonstrates that lipid nanodiscs provide a unique approach for <i>in vitro</i> studies of native membrane proteins using SMFS and open an avenue to characterize membrane proteins by a wide variety of SMFS approaches that have been established on water-soluble proteins