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 longrange 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.

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.

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.

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.

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.