Biotin–dendrimer conjugates (such as biotin-PAMAMs-NH2) are important macromolecules in the field of host–guest chemistry and widely used systems for delivery. The similar chemical structures of the inner and outer layers of PAMAM-NH2 make it difficult to illuminate the interaction and the binding affinity of biotin-PAMAMs-NH2. By utilizing NMR techniques including 1H NMR titration, CSSF-TOCSY, STDD methods, and 2D DOSY analysis, we demonstrate a method to sort out these interactions. The methylene protons of the inner and outer layers of PAMAM-NH2 are successfully identified and accurately positioned so that the carboxylic acid groups of biotins are having ionic interactions with the outermost amine groups of PAMAM-NH2. The inner PAMAM-NH2 is protonated when reaching the isoelectric point of PAMAM-NH2, increasing the hydrodynamic radius. On the basis of the NMR experiments, a model is proposed, where the carboxylic acid groups and heterocyclic skeleton of biotin arched over the outer layers of PAMAM-NH2 like a bridge. Furthermore, using STDD epitope mapping, the binding affinity between biotin and PAMAM-NH2 was quantified. The diffusion behavior of biotin-G5 PAMAM-NH2 complex is more complicated than that of biotin-G3 PAMAM-NH2 complex due to steric hindrance. The results provide a theoretical basis for understanding these complicated drug delivery systems.
Yang Yanwu,Glidden Michael D.,Dhayalan Balamurugan,Zaykov Alexander N.,Chen Yen-Shan,Wickramasinghe Nalinda P.,DiMarchi Richard D.,Weiss Michael A.
Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome’s variable phenotypic spectrum—onsets ranging from the neonatal period to later in childhood or adolescence—reflects structural features of respective folding intermediates.
危险品化学品经营许可证（带存储）