Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors

HIV-1 Nef perturbs artificial membranes: investigation of the contribution of the myristoyl anchor and of the basic amino acid cluster

Ziel der vorliegenden Arbeit war die Untersuchung der Interaktion des HIV-1 Nef Pro-teins mit artifiziellen Membransystemen. In der Literatur wurde in den 1990ern postu-liert, dass der N-terminale Lipidanker für die Wechselwirkung des Proteins mit Membra-nen essentiell ist. Allerdings wurden in letzter Zeit Studien veröffentlicht, die zeigen, dass auch das unmyristoylierte Protein mit Membranen assoziiert sein kann. Somit muss es noch andere Strukturmotive geben, die die Wechselwirkung maßgeblich beeinflussen. Daher wurde in dieser Arbeit zum einen der Einfluss des N-terminalen Myristoylankers untersucht und zum anderen der Einfluss des positiven Ladungsclusters im N-Terminus des Proteins. Hierzu wurden neben den Messungen mit wt Nef auch Messungen mit der unmyristoylierten Mutante Nef G2A sowie mit Mutanten mit reduzierter positiver La-dungsdichte Nef KKAA und Nef R4A durchgeführt. Überraschenderweise konnte sowohl bei Experimenten an unilamellaren Lipidvesikeln als auch bei Experimenten mit festkörperunterstützten Lipiddoppelschichten keine Bindung des Proteins an Membranen beobachtet werden. Vielmehr lassen die Untersuchungen darauf schließen, dass das Protein in der Lage ist, die Stabilität von Lipid-doppelschichten zu stören und Lipide aus dem Membranverbund herauszulösen. Dies zeigte sich bei den Release-Messungen in einer Freisetzung des Farbstoffs 5(6)-Carboxyfluorescein. Bei den QCM-Experimenten konnte eine Anstieg der Resonanzfrequenz der Schwingquarzes und bei den Ellipsometriemessungen eine Verringe-rung der Schichtdicke des Membransystems beobachtet werden. Mittels der hochauflösenden bildgebenden Methoden Fluoreszenz- und Rasterkraftmikroskopie konnte diese Störung der Struktur von Lipiddoppelschichten visualisiert werden. Durch den Einsatz eines FITC-konjugierten Antikörpers konnte gezeigt werden, dass HIV-1 Nef in den ge-störten Bereichen der Membran lokalisiert ist. Mittels rasterkraftmikroskopischer Unter-suchungen konnte das Protein ebenfalls in den Membrandefekten visualisiert werden. Abgeseh en von den Release-Messungen mit der Lipidmischung POPC:POPS (4:1) zeigte sich bei den Experimenten, dass der N-terminale Lipidanker des Proteins die Interaktion mit artifiziellen Membranen nicht maßgeblich beeinflusst. Sowohl an Lipidvesikeln als auch an festkörperunterstützten Membranen lagen die von Nef G2A induzierten Störun-gen in der Organisation der Lipiddoppelschichten in der gleichen Größenordnung wie die von wt Nef verursachten. Eine Verringerung des positiven Ladungsclusters im N-Terminus des Proteins beeinflusst die Wechselwirkung mit Membranen stark. Dies zeigten die Untersuchungen mit den Mutanten Nef KKAA und Nef R4A. Diese Mutanten destabilisierten die untersuchten Membransysteme in viel geringerem Umfang als wt Nef. Die Mutanten Nef KKAA führte dabei noch zu einer etwas größeren Störung der Mem-branstruktur als Nef R4A. Dies könnte zum einen darin begründet sein, dass Nef KKAA im Gegensatz zu Nef R4A mit nur noch 3 positiven Ladungen noch 5 positive Ladungen besitzt, zum anderen könnte es auch zeigen, dass die N-terminalen Arginine eine wichtigere Rolle bei der Wechselwirkung mit Membranen spielen als die Lysine. In Rahmen dieser Arbeit wurde ebenfalls der Einfluss der Lipidzusammensetzung von Membranen untersucht. Hierbei zeigte sich, dass das Einbringen von negativer Ladungsdichte in die Membranen zu einer Verringerung der Aktivität des Proteins führt. Allerdings scheint vor allem bei wt Nef mehr die Struktur der Kopfgruppe des Lipids die Interaktion zu beeinflussen als die Ladung selbst

Nanostructured Carbonaceous Materials from Molecular Precursors

Nanostructured carbonaceous materials, that is, carbon materials with a feature size on the nanometer scale and, in some cases, functionalized surfaces, already play an important role in a wide range of emerging fields, such as the search for novel energy sources, efficient energy storage, sustainable chemical technology, as well as organic electronic materials. Furthermore, such materials might offer solutions to the challenges associated with the on-going depletion of nonrenewable energy resources or climate change, and they may promote further breakthroughs in the field of microelectronics. However, novel methods for their preparation will be required that afford functional carbon materials with controlled surface chemistry, mesoscopic morphology, and microstructure. A highly promising approach for the synthesis of such materials is based on the use of well-defined molecular precursors. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Multi-Set Point Intermittent Contact (MUSIC) Mode Atomic Force Microscopy of Oligothiophene Fibrils

We developed MUSIC-mode atomic force microscopy (AFM) to emulate intermittent contact mode AFM without a feedback loop and in the absence of lateral forces. This single-pass approach is based On maps of amplitude-phase-distance curves and allows the height and to phase images to be simultaneously obtained for almost any amplitude set point. This is advantageous for determining the shape and nanomechanical properties of very soft and fragile samples. As an example, we studied supramolecular aggregates of oligothiophenes, which form approximate to 15-nm wide fibrils with a rigid core and a soft shell

HIV-1 Nef Perturbs Artificial Membranes: Investigation of the Contribution of the Myristoyl Anchor

Nef, an accessory protein from human immunodeficiency virus type 1, is critical for optimal viral replication and pathogenesis. Here, we analyzed the influence of full-length myristoylated and nonmyristoylated Nef on artificial lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By means of cosedimentation assays, we found that neither nonmyristoylated nor myristoylated Nef stably binds to POPC unilamellar vesicles. Time-resolved ellipsometry rather indicates that the proteins perturb the assembly of POPC planar bilayers. This observation was corroborated by fluorescence and scanning force microscopy, suggesting that membrane disordering occurs upon interaction of full-length myristoylated and nonmyristoylated Nef with planar POPC membranes immobilized on SiO(2) surfaces resulting in loss of material from the surface. The membrane perturbations were further investigated by vesicle release experiments, demonstrating that the disordering results in defects through which the fluorophor carboxyfluorescein can pass. From these results, we conclude that Nef is capable of disordering and perturbing lipid membranes and that the myristoyl group is not the decisive determinant for the action of the protein on lipid membranes

Facile synthesis of oligoyne amphiphiles and their rotaxanes

Carbon-rich organic compounds containing a series of conjugated triple bonds (oligoynes) are relevant synthetic targets, but an improved access to oligoynes bearing functional groups would be desirable. Here, we report the straightforward synthesis of two series of oligoyne amphiphiles with glycoside or carboxylate polar head groups, investigate their self-assembly behavior in aqueous media, and their use as precursors for the formation of oligoyne rotaxanes with cyclodextrin hosts. To this end, we employed mono-, di-, or triacetylenic building blocks that gave access to the corresponding zinc acetylides in situ and allowed for the efficient elongation of the oligoyne segment in few synthetic steps via a Negishi coupling protocol. Moreover, we show that the obtained oligoyne derivatives can be deprotected to yield the corresponding amphiphiles. Depending on their head groups, the supramolecular self-assembly of these amphiphiles gave rise to different types of carbon-rich colloidal aggregates in aqueous media. Furthermore, their amphiphilicity was exploited for the preparation of novel oligoyne cyclodextrin rotaxanes using simple host-guest chemistry in water

Development of a robust supramolecular method to prepare well-defined nanofibrils from conjugated molecules

In order to produce materials with tailored structures and functions via supramolecular self-assembly of molecular precursors in a predictable fashion, it is necessary to develop 'supramolecular methods' based on structurally simple 'supramolecular synthons'. Thus, the formation of one-dimensional aggregates from pi-conjugated molecules requires a combination of non-covalent interactions that efficiently suppresses lateral aggregation, promotes one-dimensional aggregation, and is also compatible with a productive pi-pi overlap of the constituent molecules. In the present work, we demonstrate that oligopeptide-polymer derivatives comprising a flexible polymer segment terminally attached to a beta-sheet-forming oligopeptide segment are structurally simple substituents that perfectly fulfill these requirements. We synthesized a matrix of diacetylene model compounds that carried oligopeptide-polymer substituents with varying degrees of polymerization of the attached polymers and different length oligopeptide segments. We combined solution-phase IR spectroscopy, AFM imaging and the topochemical diacetylene polymerization as a highly sensitive probe for the molecular arrangement and the degree of order inside aggregates obtained in organic solvents. The thus determined molecular parameters for the reliable formation of well-defined nanoscopic fibrillar structures with uniform diameters, and defined helical 'core-shell' morphologies were then successfully transferred to analogous perylene bisimide and quaterthiophene derivatives, demonstrating the versatility and robustness of the chosen molecular design

Multi-Set Point Intermittent Contact (MUSIC) Mode Atomic Force Microscopy of Oligothiophene Fibrils

We developed MUSIC-mode atomic force microscopy (AFM) to emulate intermittent contact mode AFM without a feedback loop and in the absence of lateral forces. This single-pass approach is based on maps of amplitude-phase-distance curves and allows the height and phase images to be simultaneously obtained for almost any amplitude set point. This is advantageous for determining the shape and nanomechanical properties of very soft and fragile samples. As an example, we studied supramolecular aggregates of oligothiophenes, which form ≈15 nm wide fibrils with a rigid core and a soft shell

Low-Temperature Preparation of Tailored Carbon Nanostructures in Water

The development of low-temperature carbonization procedures promises to provide novel nanostructured carbon materials that are of high current interest in materials science and technology. Here, we report a "wet-chemical" carbonization method that utilizes hexayne amphiphiles as metastable carbon precursors. Nearly perfect control of the nanoscopic morphology was achieved by self-assembly of the precursors into colloidal aggregates with tailored diameter in water. Subsequent carbonization furnished carbon nanocapsules with a carbon microstructure resembling graphite-like amorphous carbon materials

Hierarchically Structured Microfibers of ‘Single Stack’ Perylene Bisimide and Quaterthiophene Nanowires

Organic nanowires and microfibers are excellent model systems for charge transport in organic semiconductors under nanoscopic confinement and may be relevant for future nanoelectronic devices. For this purpose, however, the preparation of well-ordered organic nanowires with uniform lateral dimensions remains a challenge to achieve. Here, we used the self-assembly of oligopeptide-substituted perylene bisimides and quaterthiophenes to obtain ‘well-ordered’ nanofibrils. The individual nanofibrils were investigated by spectroscopic and imaging methods, and the preparation of hierarchically structured microfibers of aligned nanofibrils allowed for a comprehensive structural characterization on all length scales with molecular level precision. Thus, we showed that the molecular chirality resulted in supramolecular helicity, which apparently serves to suppress lateral aggregation so that the individual nanofibrils comprised a single stack of the π-conjugated molecules at their core. Moreover, the conformational flexibility between the hydrogen-bonded oligopeptides and the π–π stacked chromophores gave rise to synergistically enhanced strong π–π interactions and hydrogen-bonding. The result is a remarkably tight π–π stacking inside the nanofibrils, irrespective of the electronic nature of the employed chromophores, which may render them suitable nanowire models to investigate one-dimensional charge transport along defined π-π stacks of p-type or n-type semiconductors