Molecular mechanisms of steric pressure generation and membrane remodeling by disordered proteins.

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Förster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape.

WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation.

WASP-family proteins are known to promote assembly of branched actin networks by stimulating the filament-nucleating activity of the Arp2/3 complex. Here, we show that WASP-family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP-family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline-rich sequence that binds profilin-actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline-rich sequences are required to support polymerase activity by (i) bringing polymerization-competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin-actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP-family proteins that create it. Collaboration between WH2 and proline-rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP-family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.

Clathrin senses membrane curvature.

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.

A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces.

Cellular membranes are densely covered by proteins. Steric pressure generated by protein collisions plays a significant role in shaping and curving biological membranes. However, no method currently exists for measuring steric pressure at membrane surfaces. Here, we developed a sensor based on Förster resonance energy transfer (FRET), which uses the principles of polymer physics to precisely detect changes in steric pressure. The sensor consists of a polyethylene glycol chain tethered to the membrane surface. The polymer has a donor fluorophore at its free end, such that FRET with acceptor fluorophores in the membrane provides a real-time readout of polymer extension. As a demonstration of the sensor, we measured the steric pressure generated by a model protein involved in membrane bending, the N-terminal homology domain (ENTH) of Epsin1. As the membrane becomes crowded by ENTH proteins, the polymer chain extends, increasing the fluorescence lifetime of the donor. Drawing on polymer theory, we use this change in lifetime to calculate steric pressure as a function of membrane coverage by ENTH, validating theoretical equations of state. Further, we find that ENTH's ability to break up larger vesicles into smaller ones correlates with steric pressure rather than the chemistry used to attach ENTH to the membrane surface. This result addresses a long-standing question about the molecular mechanisms of membrane remodeling. More broadly, this sensor makes it possible to measure steric pressure in situ during diverse biochemical events that occur on membrane surfaces, such as membrane remodeling, ligand-receptor binding, assembly of protein complexes, and changes in membrane organization.

Membrane fission by protein crowding.

Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

Receptor Heterodimerization Modulates Endocytosis through Collaborative and Competitive Mechanisms.

Recruitment of receptors into clathrin-coated structures is essential to signal transduction and nutrient uptake. Among the many receptors involved in these processes, a significant fraction forms dimers. Dimerization of identical partners has generally been thought to promote receptor recruitment for uptake because of increased affinity of the dimer for the endocytic machinery. But what happens when receptors with substantially different affinities for the endocytic machinery come together to form a heterodimer? Evidence from diverse receptor classes, including G-protein-coupled receptors and receptor tyrosine kinases, suggests that heterodimerization with a strongly recruited receptor can drive significant recruitment of a receptor that lacks direct interactions with the endocytic machinery. However, a systematic biophysical understanding of this effect has yet to be established. Motivated by the potential of such events to influence cell signaling, here, we investigate the impact of receptor heterodimerization on endocytic recruitment using a family of engineered model receptors. As expected, we find that dimerization of a weakly recruited receptor with a strongly recruited receptor promotes incorporation of the weakly recruited receptor to endocytic structures. However, the effectiveness of this collaborative mechanism depends heavily on the relative strengths of endocytic recruitment of the two receptors that make up the dimer. Specifically, as the strength of endocytic recruitment of the weakly recruited receptor approaches that of the strongly recruited receptor, monomers of each receptor compete with heterodimers for space within endocytic structures. In this regime, the presence of the strongly recruited receptor drives a reduction in incorporation of the weakly recruited receptor into clathrin-coated structures. Similarly, as the strength of the dimer bond between the two receptors is progressively weakened, competition begins to dominate over collaboration. Collectively, these results demonstrate that the impact of receptor heterodimerization on endocytic recruitment is controlled by a delicate balance between collaborative and competitive mechanisms.

Liposome-Mediated Chemotherapeutic Delivery Is Synergistically Enhanced by Ternary Lipid Compositions and Cationic Lipids.

Most small molecule chemotherapeutics must cross one or more cellular membrane barriers to reach their biochemical targets. Owing to the relatively low solubility of chemotherapeutics in the lipid membrane environment, high doses are often required to achieve a therapeutic effect. The resulting systemic toxicity has motivated efforts to improve the efficiency of chemotherapeutic delivery to the cellular interior. Toward this end, liposomes containing lipids with cationic head groups have been shown to permeabilize cellular membranes, resulting in the more efficient release of encapsulated drugs into the cytoplasm. However, the high concentrations of cationic lipids required to achieve efficient delivery remain a key limitation, frequently resulting in toxicity. Toward overcoming this limitation, here, we investigate the ability of ternary lipid mixtures to enhance liposomal delivery. Specifically, we investigate the delivery of the chemotherapeutic, doxorubicin, using ternary liposomes that are homogeneous at physiological temperature but have the potential to undergo membrane phase separation upon contact with the cell surface. This approach, which relies upon the ability of membrane phase boundaries to promote drug release, provides a novel method for reducing the overall concentration of cationic lipids required for efficient delivery. Our results show that this approach improves the performance of doxorubicin by up to 5-fold in comparison to the delivery of the same drug by conventional liposomes. These data demonstrate that ternary lipid compositions and cationic lipids can be combined synergistically to substantially improve the efficiency of chemotherapeutic delivery in vitro.

Molecular thermodynamics of receptor competition for endocytic uptake.

Endocytic uptake of receptors from the cell surface plays an important role in diverse processes from cell signaling to nutrient internalization. Understanding the mechanisms by which endocytic structures select receptors for internalization is of fundamental importance to our understanding of cellular physiology. Binding of receptors to the endocytic protein machinery is known to facilitate receptor loading into endocytic structures. However, many receptor species use the same small set of biochemical motifs to interact with the endocytic machinery, suggesting that receptors may compete for a limited number of binding sites within endocytic structures. Previous studies have shown that such competition can substantially modify receptor uptake. However, a predictive biophysical understanding of this phenomenon is currently lacking. Toward addressing this gap, here we employ quantitative imaging and statistical thermodynamics to measure and predict the competition between two distinct receptor species that are internalized simultaneously from the cell surface. Our studies demonstrate that when receptors compete for the same interactions with the endocytic machinery, their uptake is fundamentally coupled. Importantly, we find that these trends can be quantitatively predicted by a simple thermodynamic analysis. These results suggest that multiple receptor species reach an equilibrium partitioning between endocytic structures and the surrounding plasma membrane as the receptors compete for occupancy within dynamic endocytic structures. More broadly, this work provides a quantitative framework for predicting the impact of competition on receptor uptake, an effect which has the potential to physically couple signaling pathways that impact diverse aspects of cellular physiology.

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein-protein interactions and self-association.

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.

Entropic Control of Receptor Recycling Using Engineered Ligands.

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Breaking through the false coincidence barrier in electron-ion coincidence experiments.

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ∼103 has largely precluded its use for this purpose, where a dynamic range of at least 105 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniform intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2-3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar9+, whereas Ar4+ is the largest observable cluster under traditional operation. This advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.

Designing lipids for selective partitioning into liquid ordered membrane domains.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

Lipid membrane domains for the selective adsorption and surface patterning of conjugated polyelectrolytes.

Conjugated polyelectrolytes (CPEs) are promising materials for generating optoelectronics devices under environmentally friendly processing conditions, but challenges remain to develop methods to define lateral features for improved junction interfaces and direct optoelectronic pathways. We describe here the potential to use a bottom-up approach that employs self-assembly in lipid membranes to form structures to template the selective adsorption of CPEs. Phase separation of gel phase anionic lipids and fluid phase phosphocholine lipids allowed the formation of negatively charged domain assemblies that selectively adsorb a cationic conjugated polyelectrolyte (P2). Spectroscopic studies found the adsorption of P2 to negatively charged membranes resulted in minimal structural change of the solution phase polymer but yielded an enhancement in fluorescence intensity (~50%) due to loss of quenching pathways. Fluorescence microscopy, dynamic light scattering, and AFM imaging were used to characterize the polymer-membrane interaction and the polymer-bound domain structures of the biphasic membranes. In addition to randomly formed circular gel phase domains, we also show that predefined features, such as straight lines, can be directed to form upon etched patterns on the substrate, thus providing potential routes toward the self-organization of optoelectronic architectures.

Steric confinement of proteins on lipid membranes can drive curvature and tubulation.

Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular structures including endocytic pits and filopodia. Binding of specific proteins to lipid membranes has been shown to promote membrane bending during endocytosis and transport vesicle formation. Additionally, specific lipid species are found to colocalize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for lipid domains in membrane bending. However, the specific mechanisms by which lipids and proteins collaborate to induce curvature remain unknown. Here we demonstrate a new mechanism for induction and amplification of lipid membrane curvature that relies on steric confinement of protein binding on membrane surfaces. Using giant lipid vesicles that contain domains with high affinity for his-tagged proteins, we show that protein crowding on lipid domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable buds and tubules. In contrast to previously described bending mechanisms relying on local steric interactions between proteins and lipids (i.e. helix insertion into membranes), this mechanism produces tubules whose dimensions are defined by global parameters: domain size and membrane tension. Our results suggest the intriguing possibility that confining structures, such as lipid domains and protein lattices, can amplify membrane bending by concentrating the steric interactions between bound proteins. This observation highlights a fundamental physical mechanism for initiation and control of membrane bending that may help explain how lipids and proteins collaborate to create the highly curved structures observed in vivo.

Selective endocytic uptake of targeted liposomes occurs within a narrow range of liposome diameter.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays, rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we have employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes, rather than the density of the ligands on their surfaces, primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Transmembrane coupling of liquid-like protein condensates.

Liquid-liquid phase separation of proteins occurs on both surfaces of cellular membranes during diverse physiological processes. In vitro reconstitution could provide insight into the mechanisms underlying these events. However, most existing reconstitution techniques provide access to only one membrane surface, making it difficult to probe transmembrane phenomena. To study protein phase separation simultaneously on both membrane surfaces, we developed an array of freestanding planar lipid membranes. Interestingly, we observed that liquid-like protein condensates on one side of the membrane colocalized with those on the other side, resulting in transmembrane coupling. Our results, based on lipid probe partitioning and mobility of lipids, suggest that protein condensates locally reorganize membrane lipids, a process which could be explained by multiple effects. These findings suggest a mechanism by which signals originating on one side of a biological membrane, triggered by protein phase separation, can be transferred to the opposite side.

Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Structural distributions from single-molecule measurements as a tool for molecular mechanics.

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence P(n)CG(3)K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Förster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models-including the worm-like chain, the freely jointed chain, and the self-avoiding chain-could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 Å, ~67 Å, ~51 Å, and ~76 Å for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P(8) exists as the extended all-trans isomaer whereas P(12) and P(15) predominantly contained one proline residue in the cis conformation. P(24) exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules.

Targeting proteins to liquid-ordered domains in lipid membranes.

We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.