Kraftinduzierte Sekundärstrukturänderungen in einzelnen Molekülen

In dieser Arbeit wurde die in den letzten Jahren entwickelte Technik der auf dem Kraftmikroskop basierenden Einzelmolekül-Kraftspektroskopie auf zwei spezielle Systeme angewendet: Poly(ethylen Glycol) (PEG) und das Membranprotein Bakteriorhodopsin. Im Vordergrund stand dabei die Analyse der mechanischen Stabilität von Sekundärstrukturen. PEG ist aufgrund seiner ungewöhnlich hohen Wasserlöslichkeit eines der wichtigsten Polymere mit einer Vielzahl von technischen Anwendungen. Es wurden Messungen zur Elastizität einzelner PEG in Abhängigkeit des Lösungsmittels durchgeführt. Mit diesen Messungen konnte der genaue molekulare Mechanismus nachvollzogen werden, der zu der hohen Wasserlöslichkeit führt. Zudem konnte gezeigt werden, wie mit einem einfachen Modell neben Konformationsänderungen auch Konformationsenergien des Polymers quantitativ bestimmt werden können. Die Kraftspektroskopie als Instrument zur Untersuchung von mechanisch induzierten Strukturänderungen, z.B. der Proteinentfaltung, wurde bisher immer nur auf polymere Strukturen angewendet. Ziel der Messungen an Bakteriorhodopsin war es zu zeigen, dass sich diese Technik auch auf Moleküle anwenden lässt, die nicht aus sich wiederholenden Einheiten aufgebaut sind. Es konnte der genaue Entfaltungsweg von Bakteriorhodopsin mit all seinen stabilen Zwischenstrukturen bestimmt werden. Mechanismen der Stabilisierung wie die Nachbarschaft von Helices, hydrophobe Wechselwirkung und der Einfluss räumlicher Einschränkung konnten dabei in ihren Auswirkungen auf die Stabilität der Proteinstruktur beobachtet werden

Immobilization of Homogeneous Monomeric, Oligomeric and Fibrillar Aβ Species for Reliable SPR Measurements

There is strong evidence that the amyloid-beta peptide (Aß) plays a central role in the pathogenesis of Alzheimer’s disease (AD). In this context, a detailed quantitative description of the interactions with different Aß species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aß in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aß conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aß allows reliable SPR measurements and quality control of the immobilized Aß aggregate species at any step throughout the experiment

Synergetic Antimicrobial Activity and Mechanism of Clotrimazole-Linked CO-Releasing Molecules

This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) through fellowship PD/BD/ 148006/2019 (SSM), PTDC/SAU-INF/29313/2017 grant, and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. The NMR data was acquired at CERMAX, Instituto de Tecnologia Quıḿ ica e Bioloǵ ica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/ SAICT/2016. This work was partially supported by the PPBIPortuguese Platform of BioImaging (PPBI-POCI-01- 0145-FEDER-022122) cofunded by national funds from OE “Orçamento de Estado” and by European funds from FEDER“Fundo Europeu de Desenvolvimento Regional”. LMS and SSM acknowledge funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 810856. H.B.-O., T.S., C.M., F.O., J.B., and M.A. gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434 (TRR 261, projects A01, A06, A10, and Z02). A.B. appreciates funding by the German Federal Ministry for Education and Research (project Gramneg. Design).Several metal-based carbon monoxide-releasing molecules (CORMs) are active CO donors with established antibacterial activity. Among them, CORM conjugates with azole antibiotics of type [Mn(CO)3(2,2′-bipyridyl)(azole)]+ display important synergies against several microbes. We carried out a structure-activity relationship study based upon the lead structure of [Mn(CO)3(Bpy)(Ctz)]+ by producing clotrimazole (Ctz) conjugates with varying metal and ligands. We concluded that the nature of the bidentate ligand strongly influences the bactericidal activity, with the substitution of bipyridyl by small bicyclic ligands leading to highly active clotrimazole conjugates. On the contrary, the metal did not influence the activity. We found that conjugate [Re(CO)3(Bpy)(Ctz)]+ is more than the sum of its parts: while precursor [Re(CO)3(Bpy)Br] has no antibacterial activity and clotrimazole shows only moderate minimal inhibitory concentrations, the potency of [Re(CO)3(Bpy)(Ctz)]+ is one order of magnitude higher than that of clotrimazole, and the spectrum of bacterial target species includes Gram-positive and Gram-negative bacteria. The addition of [Re(CO)3(Bpy)(Ctz)]+ to Staphylococcus aureus causes a general impact on the membrane topology, has inhibitory effects on peptidoglycan biosynthesis, and affects energy functions. The mechanism of action of this kind of CORM conjugates involves a sequence of events initiated by membrane insertion, followed by membrane disorganization, inhibition of peptidoglycan synthesis, CO release, and break down of the membrane potential. These results suggest that conjugation of CORMs to known antibiotics may produce useful structures with synergistic effects that increase the conjugate's activity relative to that of the antibiotic alone.publishersversionpublishe

Direct and Model Free Calculation of Force-Dependent Dissociation Rates from Force Spectroscopic Data

Force spectroscopy allows testing the free energy landscapes of molecular interactions. Usually, the dependency of the most probable rupture force on the force rate or the shape of the rupture force histogram is fitted with different models that contain approximations and basic assumptions. We present a simple and model free approach to extract the force-dependent dissociation rates directly from the force curve data. Simulations show that the dissociation rates at any force are given directly by the ratio of the number of detected rupture events to the time this force was acting on the bond. To calculate these total times of acting forces, all force curve data points of all curves measured are taken into account, which significantly increases the amount of information which is considered for data analysis compared to other methods. Moreover, by providing force-dependent dissociation rates this method allows direct testing and validating of any energy landscape model

Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy

Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors

Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide

Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins