Oligoglycerol Detergents for Native Mass Spectrometry of Membrane Proteins

Membrane proteins are associated with biological membranes and fulfill functions that are vital for all living organisms. They mediate many physiological processes and are therefore important targets for the development of new pharmaceutical drugs. Numerous biophysical approaches have been developed that address the elucidation of their three-dimensional structure upon isolation. In this context, detergents are important tools, because they can isolate and stabilize membrane proteins apart from their native host. In recent years there has been a growing interest in studying the structure and dynamics of isolated membrane protein complexes by native mass spectrometry (MS). This technique can provide information about the mass and composition of a membrane protein complex and allows one to study interactions with structurally relevant ligands, such as drugs, nucleotides, or lipids. Detergents that are suitable for native MS do not only require solution conditions appropriate for membrane protein isolation but also gas-phase properties that help to retain native protein structures and their non-covalent interactions in the vacuum of a MS instrument. However, detergent families that are currently available can facilitate either the isolation of membrane proteins or their analysis by native MS. This thesis addresses the question, how one can optimize the molecular structure of a detergent family for membrane protein isolation and individual applications in native MS. With this regard, the utility of dendritic oligoglycerol detergents (OGDs) was evaluated, which have not yet been used in membrane protein research. For this purpose, a highly systematic OGD library was constructed by systematically changing the structure of these detergents. In order to prove their general utility for protein MS, mixtures between soluble proteins and OGDs were analyzed by native MS. Data obtained from the soluble protein ß-lactoglobulin (BLG) indicated that protein charge states can be manipulated by tuning the basicity of these detergents. A comparative study between BLG and the membrane protein OmpF revealed the validity of this design criterion and demonstrated for the first time the utility of OGDs for native MS of membrane proteins. Moreover, with a set of five different OGDs and four membrane proteins it was examined, how crucial aspects, such as membrane protein isolation, charge states, and the ability to detect binding with structurally relevant membrane lipids could be independently optimized by altering the OGDs’ molecular structure. Furthermore, these detergents enabled the easy MS analysis of a G protein-coupled receptor protein (GPCR), which is one of the most challenging and most important protein families in pharmacology. The obtained results open new avenues for the future development of detergents that cover optimal properties for membrane protein isolation and native MS applications.Membranproteine sind ein essentieller Bestandteil von biologischen Membranen. Sie tragen maßgeblich zur Regulierung von physiologischen Prozessen bei und bilden deshalb einen wichtigen Anhaltspunkt bei der Entwicklung von neuen Medikamenten. Verschiedene biophysikalische Methoden wurden entwickelt, um die dreidimensionale Struktur von Membranproteinen zu untersuchen. In diesem Zusammenhang haben Detergenzien eine besondere Bedeutung, denn sie können dabei helfen Membranproteine unter Erhalt ihrer nativen Struktur zu isolieren. In den vergangenen Jahren wuchs das Interesse an der Strukturuntersuchung von isolierten Membranproteinen mittels nativer Massenspektrometrie (MS). Diese Technik liefert Informationen über Masse und Zusammensetzung von komplexen Membranproteinen und ermöglicht die Untersuchung von Wechselwirkungen zu wichtigen Liganden, wie z. B. Medikamenten, Nukleotiden oder Lipiden. Die in diesem Zusammenhang bisher untersuchten Detergenzien bieten entweder vorteilhafte Eigenschaften für die Isolierung von Membranproteinen oder für deren Untersuchungen mittels nativer MS. In der vorliegenden Arbeit wurde die Fragestellung adressiert, ob es möglich ist die molekulare Struktur von Detergenzien für beide Anwendungsgebiete zu optimieren. Dafür wurde erstmalig das Potential von dendritischen Oligoglycerol-Detergenzien (OGDs) evaluiert. Für diesen Zweck wurde eine Substanzbibliothek aufgebaut, indem einzelne Strukturparameter der OGDs systematisch variiert wurden. Um die generelle Anwendbarkeit von OGDs für die MS-Analyse von Proteinen zu evaluieren, wurden im nächsten Schritt Mischungen von löslichen Proteinen und OGDs mittels MS untersucht. Beobachtungen führten zu der Hypothese, dass sich Ladungszustände von Proteinen durch die Basizität der OGDs steuern lassen. Weiterführende Studien mit dem trimeren Membranprotein OmpF untermauerten diese Hypothese und bestätigten darüber hinaus die Anwendbarkeit von OGDs für die Analyse von Membranproteinen mittels nativer MS. In einer weiteren Studie wurde mit Hilfe von fünf verschiedenen OGDs und vier verschiedenen Membranproteinen gezeigt, wie sich durch Variationen in der OGD-Struktur wichtige Aspekte individuell steuern lassen, z. B. die Isolierung von Membran- proteinen, Ladungsreduktion und die Möglichkeit Komplexe zwischen Membranproteinen und Lipiden zu detektieren. Darüber hinaus ermöglichten OGDs die einfache MS-Analyse von einem G-Protein- gekoppelten Rezeptor, welche derzeit eine der am schwierigsten zu untersuchenden Proteinfamilien ist und dessen Struktur von außerordentlicher pharmakologischer Bedeutung ist. Die vorliegenden Ergebnisse zeigen, wie sich die molekulare Struktur von OGDs für die Isolierung von Membranproteinen und nativer MS-Anwendungen optimieren lässt. Darauf aufbauend lassen sich neue Detergenzien entwickeln, welche in Zukunft die Untersuchungen von Membranproteinen erleichtern können

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Detergents enable the purification of membrane proteins and are indispensable reagents instructural biology. Even though a large variety of detergents have been developed in the lastcentury, the challenge remains to identify guidelines that allowfine-tuning of detergents forindividual applications in membrane protein research. Addressing this challenge, here weintroduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS)reveals that the modular OGD architecture offers the ability to control protein purificationand to preserve interactions with native membrane lipids during purification. In addition to abroad range of bacterial membrane proteins, OGDs also enable the purification and analysisof a functional G-protein coupled receptor (GPCR). Moreover, given the modular design ofthese detergents, we anticipatefine-tuning of their properties for specific applications instructural biology. Seen from a broader perspective, this represents a significant advance forthe investigation of membrane proteins and their interactions with lipids

Online monitoring the isomerization of an azobenzene-based dendritic bolaamphiphile using ion mobility-mass spectrometry

Ion mobility-mass spectrometry was used to obtain detailed information about the kinetics of the light-induced cis/trans isomerization process of a new supramolecular azobenzene-based bolaamphiphile. Further experiments revealed that the investigated light-induced structural transition dramatically influences the aggregation behaviour of the molecule

Switchable synchronisation of pirouetting motions in a redox-active [3]rotaxane

In this study, the crown/ammonium [3]rotaxane R2 is reported which allows a switchable synchronisation of wheel pirouetting motions. The rotaxane is composed of a dumbbell-shaped axle molecule with two mechanically interlocked macrocycles which are decorated with a redox-active tetrathiafulvalene (TTF) unit. Electrochemical, spectroscopic, and electron paramagnetic resonance experiments reveal that rotaxane R2 can be reversibly switched between four stable oxidation states (R2, R2˙+, R22(˙+), and R24+). The oxidations enable non-covalent, cofacial interactions between the TTF units in each state—including a stabilised mixed-valence (TTF2)˙+ and a radical-cation (TTF˙+)2 dimer interaction—which dictate a syn (R2, R2˙+, and R22(˙+)) or anti (R24+) ground state co-conformation of the wheels in the rotaxane. Furthermore, the strength of these wheel–wheel interactions varies with the oxidation state, and thus electrochemical switching allows a controllable synchronisation of the wheels’ pirouetting motions. DFT calculations explore the potential energy surface of the counter-rotation of the two interacting wheels in all oxidation states. The controlled coupling of pirouetting motions in rotaxanes can lead to novel molecular gearing systems which transmit rotational motion by switchable non-covalent interactions

Therapeutic subthalamic nucleus deep brain stimulation reverses cortico-thalamic coupling during voluntary movements in Parkinson's disease.

Deep brain stimulation of the subthalamic nucleus (STN DBS) has become an accepted treatment for patients experiencing the motor complications of Parkinson's disease (PD). While its successes are becoming increasingly apparent, the mechanisms underlying its action remain unclear. Multiple studies using radiotracer-based imaging have investigated DBS-induced regional changes in neural activity. However, little is known about the effect of DBS on connectivity within neural networks; in other words, whether DBS impacts upon functional integration of specialized regions of cortex. In this work, we report the first findings of fMRI in 10 subjects with PD and fully implanted DBS hardware receiving efficacious stimulation. Despite the technical demands associated with the safe acquisition of fMRI data from patients with implanted hardware, robust activation changes were identified in the insula cortex and thalamus in response to therapeutic STN DBS. We then quantified the neuromodulatory effects of DBS and compared sixteen dynamic causal models of effective connectivity between the two identified nodes. Using Bayesian model comparison, we found unequivocal evidence for the modulation of extrinsic (between region), i.e. cortico-thalamic and thalamo-cortical connections. Using Bayesian model parameter averaging we found that during voluntary movements, DBS reversed the effective connectivity between regions of the cortex and thalamus. This casts the therapeutic effects of DBS in a fundamentally new light, emphasising a role in changing distributed cortico-subcortical interactions. We conclude that STN DBS does impact upon the effective connectivity between the cortex and thalamus by changing their sensitivities to extrinsic afferents. Furthermore, we confirm that fMRI is both feasible and is tolerated well by these patients provided strict safety measures are adhered to

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated

Study of the Stabilization to the Nanometer Level of Mechanical Vibrations of the CLIC Main Beam

Original publication available at http://www.jacow.org/International audienceTo reach the design luminosity of CLIC, the movements of the quadrupoles should be limited to the nanometre level in order to limit the beam size and emittance growth. Below 1 Hz, the movements of the main beam quadrupoles will be corrected by a beambased feedback. But above 1 Hz, the quadrupoles should be mechanically stabilized. A collaboration effort is ongoing between several institutes to study the feasibility of the "nanostabilization" of the CLIC quadrupoles. The study described in this paper covers the characterization of independent measuring techniques including optical methods to detect nanometre sized displacements and analyze the vibrations. Actuators and feedback algorithms for sub-nanometre movements of magnets with a mass of more than 400 kg are being developed and tested. Input is given to the design of the quadrupole magnets, the supports and alignment system in order to limit the amplification of the vibration sources at resonant frequencies. A full scale mock-up integrating all these features is presently under design. Finally, a series of experiments in accelerator environments should demonstrate the feasibility of the nanometre stabilization

The Muon Anomalous Magnetic Moment and the Standard Model

The muon anomalous magnetic moment measurement, when compared with theory, can be used to test many extensions to the standard model. The most recent measurement made by the Brookhaven E821 Collaboration reduces the uncertainty on the world average of a_mu to 0.7 ppm, comparable in precision to theory. This paper describes the experiment and the current theoretical efforts to establish a correct standard model reference value for the muon anomaly.Comment: Plenary Talk; PANIC'02 XVI Particles and Nuclear International Conference, Osaka, Japan; Sept. 30 - Oct. 4, 2002; Report describes the published 0.7 ppm result and updates the theory statu