Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the tripleresonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13 C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, C α , C β , C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly nativelike topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior. (7), and the structure of a GPCRarrestin complex was solved (8).While our knowledge of the structure of GPCRs in various states and their mode of activation and desensitization is rapidly increasing, detailed information on their folding pathways is still lacking. The popular refined two-stage model from Popot and Engelman postulates that secondary structure forms when the peptide chain partitions into the membranewater interface (9-11). However, proteins destined for membrane insertion are generally subjected to the concerted action of translating ribosomes in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of eukaryotes or in the plasma membrane of bacteria (12). In order to traffic proteins to membranes in most cells the signal-recognition particle targets the nascent chains emerging from the ribosome tunnel to the translocon complex (13 (12,(18)(19)(20). Folding of polytopic membrane proteins is the result of a series of events that include helixinsertion into the hydrophobic core and sequestering of loop-sequences into cytosolic or extracellular space (11,21). The timing of the chain insertion and the localization of TM helices would be expected to be a consequence of the amino acid sequence and the interaction of the growing polypeptide chain with the membrane. Recently, however, the first evidence was obtained that helices might change their locat...

The impact of stereochemical purity of lipids on their selfassembly behavior is critical for establishing their true phase behavior from their commercial counterparts, which often contains stereoisomeric mixtures and other impurities. Here, stereochemically pure phytantriol (PT), (3,7,11,15-tetramethylhexadecane-1,2,3-triol) was synthesized from the natural trans-phytol and its thermotropic and lyotropic phase behavior in water investigated by small-angle Xray scattering (SAXS), polarized optical microscopy (POM), and differential scanning calorimetry (DSC). These chemically pure lipids contain two chiral centers at the hydrophilic head group region and two chiral centers at the lipophilic tail region, allowing us to address the question of whether the molecular stereochemistry is related to the macroscopic phase behavior of phytantriol. In contrast to its commercial stereoisomeric mixtures, which form an isotropic micellar phase, neat (2S,3S,7R,11R)-3,7,11,15-tetramethylhexadecane-1,2,3-triol (S,S-PT) shows a smectic lamellar phase at room temperature, whereas (2R,3R,7R,11R)-3,7,11,15-tetramethylhexadecane-1,2,3-triol (R,R-PT) forms solid crystals. The lyotropic phase behavior of R,R-PT appears to be identical to that of the previously reported commercial stereoisomeric PT mixtures. In contrast, S,S-PT exhibits a different phase behavior. A lamellar crystalline phase (L c ) is formed instead of an isotropic micellar phase at a low water content, which also coexisted with other phases at low temperature. Subtle change in the shape of the diastereomers leads to variable steric interactions and subsequently affects the packing of the lipids at the molecular level, thereby influencing its self-assembling behavior. Finally, lipidic cubic phase crystallization of the membrane protein bacteriorhodopsin yielded a larger number of microcrystals with a higher average crystal length from S,S-PT than from commercial PT, suggesting faster nucleation.