Structure of HsFpn in complex with 11F9 Fab.

(a) Cryo-EM density map of HsFpn in complex with 11F9 Fab in the presence of Co2+. Densities for the NTD, CTD, and Fab are colored in pale green, light orange, and slate gray, respectively and contoured at 8.5σ. (b) Cartoon representation of HsFpn-11F9 complex. Ligand residues of the transition metal ion binding sites, S1 and S2, are highlighted and shown as sticks. Co2+ is rendered as a sphere (light pink). Densities for S1 and S2 are contoured at 4σ as blue mesh. CTD, C-terminal domain; cryo-EM, cryo-electron microscopy; Fab, fragment of antigen-binding; NTD, N-terminal domain.</p

Binding affinities of PR73 to Fpn measured by Octet BLI.

Related to Fig 6. (DOCX)</p

Inhibition of PR73 against the Co2+ transport by Fpn.

Related to Fig 7. (a) Co2+ import-induced H+ export by Fpn in HEK cells indicated by fluorescence change (F/F0) of a pH-sensitive dye (pHrodo Red) loaded inside cells. A total of 500 μM of Co2+ was administered at time zero. The solid lines represent the mean of 4 repeats and the shaded areas the SD. (b) Fpn-specific pH changes from data in (a). Each bar represents the change in fluorescence after subtraction of the fluorescence change in empty vector control cells. Data is plotted as means (n = 3) with error bars representing the SEM. Unpaired Student’s t test, p = 0.0052. Source data for (a–b) can be found in S1 Data. (TIF)</p

Structural changes induced by PR73.

Structure of HsFpn-PR73 (pale green) aligned with apo-HsFpn (gray, PDB ID 6W4S) viewed from the side (a) and the extracellular side (b). Large structural changes are highlighted by arrows. Structures of HsFpn-PR73 aligned with HsFpn-Hepcidin (light blue, PDB ID 6WIK) viewed from the side (c) and extracellular side (d).</p

Cryo-EM analysis of HsFpn-Co2+ in nanodisc.

Related to Fig 2. (a) Representative electron micrograph (upper panel) and 2D class averages (lower panel). (b) Workflow of data processing for single-particle reconstruction. (c) The gold-standard Fourier shell correlation (FSC) curves for the final map (left panel) and map-to-model FSC curves (right panel). (d) Direction distribution of particles used in the final 3D reconstruction. (e) Local resolution map colored from 2.4 Å (blue) to >4.0 Å (red). Source data for (c) can be found in S1 Data. (TIF)</p

Stable, Compact, Bright Biofunctional Quantum Dots with Improved Peptide Coating

We developed a new peptide, natural phytochelatin (PC), which tightly binds to CdSe/ZnS quantum dots’ (QDs) surfaces and renders them water-soluble. Coating QDs with this flexible and all-hydrophilic peptide offers high colloidal stability, adds only 0.8–0.9 nm to the radius of the particles (as compared to their original inorganic radius), preserves very high quantum yield (QY) in water, and affords facile bioconjugation with various functional groups. We demonstrate specific targeting (with minimal nonspecific binding) of such fluorescein-conjugated QDs to ScFv-fused mouse prion protein expressed in live N2A cells. We also demonstrated homogeneous in vivo biodistribution with no significant toxicity in live zebrafish

Analysis of Viral Fitness and Drug Resistance.

<p>To determine if N126K is acting as a compensatory mutation in RC-101, viral fitness in culture over a 7 day period was determined for wild-type (WT) and drug-resistant mutants in the presence or absence of RC-101 and ENF. An MOI of 0.003, as determined by TCID50, was used for the initial inoculation of PM1 cells. Fitness was determined by p24 concentration measured in cell supernatants. Error bars represent SEM. Black lines indicate comparisons between conditions (N = 3; * = <i>p</i><0.05).</p

Synthesis and Characterization of BaL <i>env</i> Molecular Clones.

<p>To study the effect of gp41 mutations identified in the BaL envelope, the dominant <i>env</i> genotypes from untreated and RC-101-passaged virus were used to generate the pNBaL molecular clone containing the complete BaL envelope within pNL43 (<b>A</b>). Molecular clones possessing the envelope proteins cloned from untreated BaL (WT) or from RC-101-passaged BaL (RCres) were treated with either RC-101 (<b>B</b>) or ENF (<b>C</b>). Error bars represent SEM. Differences in percent inhibition were determined between WT and RCres at each drug concentration (N = 3; ** = <i>p</i><0.01, *** = <i>p</i><0.001).</p

Effect of HR1 and HR2 Mutations on Cell-Cell Fusion.

<p>Cell-cell fusion was analyzed to determine differences in gp41 fusion demonstrated by wild type (WT) and drug-resistant mutant virus. Transformed 293T cells expressing BaL Env and Tat proteins, as well as GFP, were co-cultured with TZM-bl reporter cells. RLU values correlate with cell-cell fusion during co-culture (<b>A</b>), which is also shown as percent fusion of WT (<b>B</b>). Error bars represent SEM. Fusion for each mutant virus was compared to that observed in WT (N = 4; or 5 * = <i>p</i><0.05, ** = <i>p</i><0.01). Fluorescent imaging and DIC were used to observe syncytia formation as a secondary indicator to compare cell-cell fusion between mutants (<b>C</b>).</p

Entry Kinetics of RC-101-Resistant Virus.

<p>Viral entry was observed over one hour to determine differences in entry kinetics between wild type (WT) and RC-101-resistant virus. Equal concentrations of infectious virus were used to infect reporter cells and infection was halted at specific time-points. Entry kinetics is shown as percent of total entry and graphed using cubic splines. Linear regression curves were fit to data and slopes were compared between the 15 and 30-minute time intervals. Error bars represent SEM. Both wild type (WT) and Q66R+N126K had significantly greater slopes than the Q66R mutant (N = 4; <i>p</i><0.05).</p