Abstract

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr³-octreotate by chemoselective oxime ligation and Cu^I-catalyzed azide–alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.

The N-terminal domain of VAT (Valosine-containing protein-like ATPase of Thermoplasma acidophilum), VAT-N (20.5 kDa), is considered to be the primary substrate-recognition site of the complex. The solution structure of VAT-N derived in our laboratory using conventionally obtained NMR restraints shows the existence of two equally sized sub-domains, VAT-Nn and VAT-Nc, together forming a kidney-shaped overall structure. The putative substrate-binding site of VAT-N involves free loops and a highly charged groove located on the surface of the protein. Alternatively, the opening of the cleft between the domains to accommodate substrate has been proposed to be part of the functional mechanism. We have used the residual dipolar couplings (RDCs) obtained in a bicelle medium to refine the structure of VAT-N. The long-range information available from RDCs both defines the sub-domain orientation and probes possible inter-domain motions. In addition, ¹⁵N backbone relaxation data were obtained and analysed within the model-free framework. Together, the data provides a refined structure with improved local geometry, but with the overall kidney shape intact. Further, the protein is rigid overall, with no evidence of inter-domain motions. Copyright © 2006 John Wiley & Sons, Ltd.

Coating of artificial surfaces with RGD (= arginine-glycine-aspartate) peptides to enhance cell adhesion is an ongoing issue. Thereby, the physiological adhesion process to the extra-cellular matrix (ECM) is mimicked by the peptide coating, leading to a strong cell-surface contact, followed by spreading and proliferation of the cells. For comparable cell adhesion studies, it is important to know the density of the RGD peptides on the surface. Here, we present an approach to determine the amount of bound cyclic RGD peptide by radio labeling with ¹²⁵I of a tyrosine-containing RGD peptide on different materials surfaces (poly(methyl methacrylate) (PMMA), titanium, and silicon). For all surfaces, the amount of bound peptides is in the range of pmol/cm¹.

Protein interactions and aggregation phenomena are probably amongst the most ubiquitous types of interactions in biological systems; they play a key role in many cellular processes. The ability to identify weak intermolecular interactions is a unique feature of NMR spectroscopy. In recent years, pulsed-field gradient NMR spectroscopy has become a convenient method to study molecular diffusion in solution. Since the diffusion coefficient of a certain molecule under given conditions correlates with its effective molecular weight, size, and shape, it is evident that diffusion can be used to map intermolecular interactions or aggregation events. Complex models can be derived from comparison of experimental diffusion data with those predicted by hydrodynamic simulations. In this review, we will give an introduction to pulsed-field gradient NMR spectroscopy and the hydrodynamic properties of proteins and peptides. Furthermore, we show examples for applying these techniques to a helical peptide and its hydrophobic oligomerization, as well as to the dimerization behavior and folding of p53.

New developments concerning alignment media for apolar solvents like chloroform make it possible to measure anisotropic parameters such as residual dipolar couplings (RDCs) at relatively low concentrations and natural isotopic abundance. As RDCs provide structural restraints with respect to an external coordinate system, long-range structural arrangements of the time-averaged structure can be determined with high precision. The method is demonstrated on the well-studied cyclo-undecapeptide Cyclosporin A (CsA), for which crystal and conventionally derived NMR structures are available. Neither crystal nor NMR structure are consistent with heteronuclear D_CHRDCs measured in a stretched poly(dimethylsiloxane) gel, and refinement by using the anisotropic parameter results in a highly defined structure with a slightly changed backbone conformation. The applied methods and interpretation of the structural model are discussed.

Partial alignment of molecules in polar organic solvents for the measurement of residual dipolar couplings (RDCs) in high-resolution NMR spectroscopy can be achieved by stretched poly(vinyl acetate) gels. The method opens the door of RDC-derived structural information (e.g. from D_CH couplings, see picture) to new classes of molecules by closing the existing gap between apolar and aqueous solutions.

Mutations in the transcription factor p53 enhance one's predisposition to cancer. Mutational studies show that double salt bridges are key elements for the dimerization of p53, its cooperative binding to DNA, and thus its proper function as a tumor suppressor (see picture). This might explain certain germ-line mutations found in patients with Li–Fraumeni syndrome.

Mutationen im Transkriptionsfaktor p53 führen zu einer erhöhten Krebsneigung. Durch Mutationsstudien wird gezeigt, dass die Bildung einer doppelten Salzbrücke entscheidend für die dimere und kooperative Bindung von DNA und damit für eine ordnungsgemäße Funktion des Tumorsuppressors ist (siehe Bild). Dies kann bestimmte Keimbahnmutationen erklären, die bei Patienten mit Li-Fraumeni-Syndrom gefunden werden.

Ein gestrecktes Polystyrol-Gel erzeugt eine partielle Orientierung von Molekülen in organischen Lösungsmitteln zur Bestimmung von dipolaren Restkopplungen (RDCs) in der NMR-Spektroskopie. Durch unterschiedliche Vernetzung im Polymer lässt sich das Ausmaß der Orientierung über einen weiten Bereich einstellen. So wird ein wichtiger NMR-Parameter durch eine Routinemessung zugänglich.

Stretched polystyrene gel (see picture) induces partial alignment of small molecules in an organic solvent for the measurement of residual dipolar couplings in high-resolution NMR spectroscopy. The degree of orientation can be varied over a wide range by the amount of cross-linking of the polymer. An important NMR parameter can thus be measured routinely.

The αvβ3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging αvβ3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.

Integrins are pivotal proteins in cell–cell adhesion, signaling and apoptosis. These properties render them attractive targets for drugs, especially those involved in cancer treatment. Recently, the structures of the extracellular domains of one of the integrin subtypes was solved with X-ray crystallography in the free form as well as bound to a ligand. These structures in combination with NMR spectroscopic data, electron microscopy images, and molecular modeling provide deeper insight into the mechanism of integrin-mediated signal transduction. The structures make structure-based rational drug design possible and are certainly hallmarks in integrin research.

Integrine sind Schlüsselproteine bei der Zell-Zell- und Zell-Matrix-Adhäsion. Sie induzieren Signaltransduktion durch die Zellmembran und sind an vielen biologischen Prozessen wie der Regulation der Migration, Zellproliferation und Apoptose (natürlicher Zelltod) beteiligt. Diese Eigenschaften machen Integrine zu attraktiven Zielproteinen für Medikamente, besonders da Integrine beim Krebswachstum eine wesentliche Rolle spielen. Kürzlich wurde die Struktur der extrazellulären Domänen eines Integrin-Subtyps ohne und mit Ligand veröffentlicht. Diese Strukturen in Kombination mit elektronenmikroskopischen Bildern, NMR-Daten und theoretischen Molekül-Modellen führen zu einem besseren Verständnis der Signaltransduktion durch Integrine. Struktur-gestütztes Liganden-Design ist nun möglich.