No abstract

Progress in colloidal synthesis in the last two decades has enabled high-quality semiconductor, plasmonic, and magnetic nanocrystals (NCs). As synthesized, these NCs are usually capped with long-chain apolar ligands. Postsynthetic surface functionalization is required for rendering such NCs colloidally stable in polar media such as water. However, unlike small anionic molecules and polymeric coatings, producing positively charged stable NCs, especially at high ionic strengths, has remained challenging. Here, we present a general approach to achieve aqueously stable cationic NCs using a set of small (<2.5 nm long) positively charged ligands. The applicability of this method is demonstrated for a variety of materials including semiconductor CdSe/CdS core/shell NCs, magnetic Fe@Fe3O4, Fe3O4, and FePt NCs, and three different classes of plasmonic Au NCs including large nanorods. The obtained cationic NCs typically have zeta potential values ranging from +30 to +60 mV and retain colloidal stability for days to months, depending on NC/ligand pair, in several biological buffers at elevated pH and in concentrated salt solutions. This allowed us to demonstrate site-specific staining of cellular structures using fluorescent cationic NCs with several different surface chemistries. Furthermore, colloidal stability of the obtained NCs in the presence of other charged species allowed the assembly of cationic and anionic counterparts driven primarily by electrostatic attraction. With this approach, we prepare highly uniform 3D and 2D binary mixtures of NCs through induced homogeneous aggregation and alternating-charge layer-by-layer deposition, respectively. Such binary mixtures may provide a new route in the engineering of nanocrystalline solids for electronics, thermoelectrics, and photovoltaics.

Cyanophycin is a biopolymer composed of long chains of β-Asp-Arg. It is widespread in nature, being synthesized by many clades of bacteria, which use it as a cellular reservoir of nitrogen, carbon, and energy. Two enzymes are known to produce cyanophycin: cyanophycin synthetase 1 (CphA1), which builds cyanophycin from the amino acids Asp and Arg by alternating between two separate reactions for backbone extension and side chain modification, and cyanophycin synthetase 2 (CphA2), which polymerizes β-Asp-Arg dipeptides. CphA2 is evolutionarily related to CphA1, but questions about CphA2’s altered structure and function remain unresolved. Cyanophycin and related molecules have drawn interest as green biopolymers. Because it only has a single active site, CphA2 could be more useful than CphA1 for biotechnological applications seeking to produce modified cyanophycin. In this study, we report biochemical assays on nine cyanobacterial CphA2 enzymes and report the crystal structure of CphA2 from Gloeothece citriformis at 3.0 Å resolution. The structure reveals a homodimeric, three-domain architecture. One domain harbors the polymerization active site and the two other domains have structural roles. The structure and biochemical assays explain how CphA2 binds and polymerizes β-Asp-Arg and highlights differences in in vitro oligomerization and activity between CphA2 enzymes. Using the structure and distinct activity profile as a guide, we introduced a single point mutation that converted Gloeothece citriformis CphA2 from a primer-dependent enzyme into a primer-independent enzyme.

Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human β2-microglobulin (β2m) and the 99-residue full-length protein. For the dimeric [20-41]β2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length β2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-β2m amyloids. Furthermore, the full-length d-enantiomer of β2m was found to be an efficient inhibitor of l-β2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.