Baoxu Liu

Trapping single molecules in liposomes: surface interactions and freeze-thaw effects.

We report on an improved method to encapsulate proteins and other macromolecules inside surface-tethered liposomes to reduce or eliminate environmental interference for single-molecule investigations. These lipid vesicles are large enough for the molecule to experience free diffusion but sufficiently small so that the molecule appears effectively immobile under the fluorescence microscope. Single-molecule fluorescence experiments were used to characterize this anchoring method relative to direct immobilization via biotin-streptavidin linkers. Multidimensional histograms of intensity, polarization, and lifetime revealed that molecules trapped in liposomes display a narrow distribution around a single peak, while the molecules directly immobilized on surface show highly dispersed values for all parameters. By hydrating the lipid film at low volumes, high encapsulation efficiencies can be achieved with ~10 times less biological material than previous protocols. We measured vesicle size distributions and found no significant advantage for using freeze-thaw cycles during vesicle preparation. On the contrary, the temperature jump can induce irreversible damage of fluorophores and it reduces significantly the functionality of proteins, as demonstrated on single-molecule binding experiments on STAT3. Our improved and biologically gentle molecule encapsulation protocol has a great potential for widespread applications in single-molecule fluorescence spectroscopy.

Lipogels: single-lipid-bilayer-enclosed hydrogel spheres.

We have fabricated Lipogels consisting of a single POPC lipid bilayer supported by a micrometer-sized, thermoresponsive, hydrophobically modified (HM), hydrogel sphere. The hydrogel consists of a lightly cross-linked poly(N-isopropylacrylamide) (pNIPAM) core surrounded by a highly cross-linked acrylic acid (AA)-rich p(NIPAM-co-AA) shell. The lipid bilayer was assembled by binding liposomes to HM microgels, followed by several cycles of freeze-thaw. The pNIPAM volume phase transition (VPT) at ∼32 °C was present both before and after hydrophobic modification and after lipid bilayer coating. Fluorescence studies confirmed the fusion of liposomes into a continuous single bilayer. At a temperature above the VPT, it was found that the volume decrease in the hydrogel was coupled to the appearance of highly curved obtrusions of the uncompromised lipid bilayer into the surroundings. It is anticipated that these properties of Lipogels will prove to be useful in drug delivery applications and in fundamental biophysical studies of membranes.

A Photostable, pH-Invariant Fluorescein Derivative for Single-Molecule Microscopy

We herein report the comprehensive characterization of the spectral and single-photon fluorescence properties of a recently synthesized fluorescein derivative and its biotinylated analog. The fluorophore displays significant increases in photostability compared to the known fluorescein label fluorescein isothiocyanate (FITC), as well as superb pH independence. This fluorescein variant has two readily accessible functional groups (aniline NH2 and phenol OH) that can be activated or blocked independently and can serve, for instance, as a fluorescent bridge between two different recognition motifs. Excellent single-photon counting fluorescence data demonstrates that it is also a particularly appropriate probe for single-molecule studies of biological interactions.

The effect of intrachain electrostatic repulsion on conformational disorder and dynamics of the Sic1 protein.

The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants. Both the end-to-end distance and the hydrodynamic radius decrease monotonically with increasing the salt concentration, and a rollover of the chain dimensions in high denaturant conditions is observed. The data were fit to the polyelectrolyte binding-screening model, yielding parameters such as the excluded volume of the uncharged chain and the binding constant to denaturant. An overall scaling factor of ∼1.2 was needed for fitting the data, which implies that Sic1 cannot be approximated by a random Gaussian chain. Fluorescence correlation spectroscopy reveals Sic1 structure fluctuations occurring on both fast (10-100 ns) and slow (∼10 ms) time scales, with the fast phase absent in low salt solutions. The results of this study provide direct evidence that long-range intrachain electrostatic repulsions are a significant factor for the conformational landscape of Sic1, and support the role of electrostatics in determining the overall shape and hydrodynamic properties of intrinsically disordered proteins.

Triggered instability of liposomes bound to hydrophobically modified core-shell PNIPAM hydrogel beads.

The ability to trigger a destabilization of the membrane integrity of liposomes bound to environmentally sensitive hydrophobically modified core-shell hydrogel beads is demonstrated. Hydrogel beads with a core composed of poly(N-isopropylacrylamide) lightly cross-linked with bisacrylamide (BA) (pNIPAM) and a shell composed of NIPAM highly cross-linked with BA and containing varying amounts of acrylic acid (AA) [p(NIPAM-co-AA)] undergo a volume phase transition (VPT) at approximately 32 degrees C, as determined from (1)H magic angle spinning (MAS) NMR, regardless of the AA content of the shell. When the shell was hydrophobically modified with either decylamine or tetradecylamine, binding of extruded large unilamellar vesicles (eLUVs) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was quantitative, as determined via fluorescence spectroscopy. Fluorescence microscopy showed that such bound eLUVs did not fuse. Hydrogel-bound eLUV membrane permeability was assessed using (31)P MAS NMR in the presence of the chemical shift agent praseodymium and demonstrated that only at lower degrees of hydrophobic modification of the core-shell hydrogels was eLUV membrane barrier integrity maintained when T < VPT. At a low degree of hydrophobic modification, cycling the temperature above the VPT even for short periods caused the eLUV membranes to become leaky. Hence, eLUV membrane permeability was coupled to the hydrogel VPT, a situation that would be useful in applications requiring triggered release of liposomal contents.

Liposome−Hydrogel Bead Complexes Prepared via Biotin−Avidin Conjugation

Liposomes immobilized onto polymeric hydrogel microbeads have potential advantages both in tissue engineering applications and as drug delivery vehicles. Here we demonstrate, quantify, and optimize lipid vesicle binding to polymeric hydrogel microbeads via the avidin-biotin conjugation system and characterize the stability of the resulting microgel-bound liposomes. Microgels consisting of a copolymer of N-isopropylacrylamide (NIPAM) and acrylic acid (AA), cross-linked with bis-acrylamide, that is, p(NIPAM-co-AA), were biotinylated using aqueous carbodiimide chemistry. Extruded liposomes consisting of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) plus a small fraction of a biotin-derivatized phosphatidylethanolamine (B-PE) were saturated with avidin and allowed to bind to biotinylated hydrogel beads. Using a combination of fluorescence spectroscopy, quenching, and microscopy and 31P NMR static and magic angle spinning (MAS) spectroscopies, we demonstrate conditions for near-quantitative liposome binding to p(NIPAM-co-AA) microbeads and show that liposome fusion does not occur under such conditions, that the liposomes remain intact and impermeable when so bound, and that they can function as slow release vehicles for entrapped aqueous species.

On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to μM range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.

Development of methods to study the conformational dynamics of quantum dot-oligonucleotide conjugates by single molecule spectroscopy

The optical properties and significant surface area of CdSe/ZnS QDs make such nanoparticles an interesting platform for the preparation of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Interactions between QDs and oligonucleotides affect biosensor performance and are not fully understood. Ensemble data obtained via FRET experiments indicated that, on average, 4-5 added oligonucleotides saturated the surface of green emitting QDs. An increase in the number of oligonucleotides per QD appeared to cause the oligonucleotides to transition from collapsed to upright conformations. Since bulk averaging hides details of such processes, methods must be developed and materials identified for studying QD-oligonucleotide conjugates at the single molecule level. Single QDs have been immobilized and fluorescence intensity trajectories measured. High count rates and good photostability were achieved using carboxyl polymer-coated QDs. Modeling of FRET efficiency based on the dimensions of QDs and oligonucleotides indicated that transitions between collapsed and upright conformations can be accurately measured based on changes in QD fluorescence lifetime. The ultimate goal of this work is to elucidate QD-oligonucleotide dynamics for better design and optimization of nucleic acid biosensors based on QDs.

Abstract B220: Artificially inducing protein-membrane anchorage: Introducing a new therapeutic modality.

We aim to develop an innovative therapeutic modality of inhibiting aberrant protein function through suppression of activation and/or nuclear translocation. Nature has developed prenylation, a post-translational modification that covalently attaches a hydrophobic prenyl group to a protein to facilitate protein-membrane association to the plasma membrane. We hypothesize that mimicking Nature by artificially inducing protein-membrane anchorage through the use of a rationally designed protein-membrane anchor (PMA), we can simultaneously inhibit the activation and nuclear translocation of oncogenic proteins. Our aim is to explore the therapeutic potential of the protein-membrane anchor and potentially develop a novel drug modality that can be utilized by cancer patients. Our proof-of-concept PMA was design to target signal transducer and activator of transcription 3 (Stat3) protein. Constitutively-active Stat3 directly contributes to the progression of cancer and is present in numerous human cancers. A number of studies have shown that down-regulation of this oncogene via iRNA knockdown induces cellular apoptosis. Thus, Stat3 is an attractive target for the development of potent anti-cancer therapeutics for cancer. Our proto-type PMA 1 was composed of two binding modules: a recognition motif to bind the protein and an anchor to sequester the protein complex to the membrane. The PMA was comprised of a potent Stat3 recognition sequence GpYLPQTV-NH2 covalently attached to a cholesterol membrane anchor. We tested the ability of our PMA to anchor Stat3 to the cell membrane in MDA-MB-231 breast cancer cells which are known to have constitutively-active Stat3. We immunostained these cells with membrane stain FM-4–64 (red), anti-Stat3 antibody (green) and DAPI (nucleus, blue). In the absence of PMA 1, there was strong Stat3 nuclear presence. Most excitingly, in the presence of 25 μM concentration PMA 1, we observed complete sequestration of Stat3 to the cell membrane through PMA-Stat3 association. Currently, we are designing and synthesizing more drug-like, nonphosphorylated PMAs that are less prone to metabolic degradation. We will conduct further studies to determine the biochemical and biological utility of these PMAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B220.

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.