Chain-length-dependent flexural rigidity revealed by molecular conformation distributions. The open circles are experimental data of poly-proline and the solid lines are fits to the full-width ...half-maximum of the semi-flexible chain model, from which the effective persistence lengths (lp) and apparent contour lengths (Leff) were estimated. Display omitted
► A mechanical view is an attractive alternative for predicting the behavior of complex molecules. ► We propose using structural distribution from smFRET to extract molecular mechanical properties. ► Short poly-l-proline peptides were used to experimentally illustrate this new approach. ► The effective persistence lengths were found to be size-dependent. ► This is the first experimental evidence of such behavior on the molecular level.
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 PnCG3K-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 ∼190Å, ∼67Å, ∼51Å, and ∼76Å 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 P8 exists as the extended all-trans isomaer whereas P12 and P15 predominantly contained one proline residue in the cis conformation. P24 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.
Lipid membranes composed of an iminodiacetic acid functionalized lipid, DSIDA, in a POPC matrix exhibited switchable properties via Cu2+ recognition to rapidly assemble microdomains that act as high ...affinity sites for His-tagged proteins. The microdomains demonstrated an order of magnitude enhanced affinity for the proteins compared to homogeneously functionalized POPC membranes with Ni2+-NTA DOGS or Cu2+-DOIDA, while a rapid release and restoration of the original membrane was accomplished with micromolar concentrations of EDTA.
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 (Lo) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the Lo phase containing DPIDA. In the presence of CuCl2, 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 Lo 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.
One of the major challenges in single-molecule studies is how to extract reliable information from the inevitably noisy data. Here, we demonstrate the unique capabilities of multichannel joint ...statistical analysis of multispectral time series using Föster resonance energy transfer (FRET) in single quantum dot (QD)−organic dye hybrids as a model system. The multispectral photon-by-photon registration allows model-free determination of intensity change points of the donor and acceptor channels independently. The subsequent joint analysis of these change points gives high-confidence assignments of acceptor photobleaching events despite the interference from background noise and from intermittent blinking of the QD donors and acceptors themselves. Finally, the excited-state lifetimes of donors and acceptors are calculated using the joint maximum likelihood estimation (MLE) method on the donor and acceptor decay profiles, guided by a four-state kinetics model.
Transient energy transfer mechanism in single quantum dot−dye hybrids was studied by following the evolution of the emission decay profile in addition to the ratiometric shift between the donor and ...acceptor. This study was made possible by the combined use of multiparameter spectroscopy and model-free statistical approach.
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.
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 develop 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 ENTH domain 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 breakup 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.
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