Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides, and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis, and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer in vivo, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one of the smallest of their kind (<100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a noninvasive cancer imaging and therapy alternative for patients. Here, we show, using high-resolution imaging and correlative techniques, that our PFH-NEs, when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis and as a multimodal imaging agent for photoacoustic, ultrasound, and fluorescence imaging of tumor growth.

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment.

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected by GTPγS. A complex of M2R and Gi1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of Gi1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gαi1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gαi1 was predominantly dimeric. The oligomeric nature of Gi1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gαi1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-Gi1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of Gi1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.