Dynamic correlations in lipid bilayer membranes over finite time intervals.

Recent single-molecule measurements [Schoch et al., Proc. Natl. Acad. Sci. U. S. A. 118, e2113202118 (2021)] have observed dynamic lipid-lipid correlations in membranes with submicrometer spatial resolution and submillisecond temporal resolution. While short from an instrumentation standpoint, these length and time scales remain long compared to microscopic molecular motions. Theoretical expressions are derived to infer experimentally measurable correlations from the two-body diffusion matrix appropriate for membrane-bound bodies coupled by hydrodynamic interactions. The temporal (and associated spatial) averaging resulting from finite acquisition times has the effect of washing out correlations as compared to naive predictions (i.e., the bare elements of the diffusion matrix), which would be expected to hold for instantaneous measurements. The theoretical predictions are shown to be in excellent agreement with Brownian dynamics simulations of experimental measurements. Numerical results suggest that the experimental measurement of membrane protein diffusion, in complement to lipid diffusion measurements, might help to resolve the experimental ambiguities encountered for certain black lipid membranes.

Correlated diffusion in lipid bilayers.

Lipid membranes are complex quasi-two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman-Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman-Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

Lipid diffusion in the distal and proximal leaflets of supported lipid bilayer membranes studied by single particle tracking.

Supported lipid bilayers (SLBs) have been studied extensively as simple but powerful models for cellular membranes. Yet, potential differences in the dynamics of the two leaflets of a SLB remain poorly understood. Here, using single particle tracking, we obtain a detailed picture of bilayer dynamics. We observe two clearly separate diffusing populations, fast and slow, that we associate with motion in the distal and proximal leaflets of the SLB, respectively, based on fluorescence quenching experiments. We estimate diffusion coefficients using standard techniques as well as a new method based on the blur of images due to motion. Fitting the observed diffusion coefficients to a two-leaflet membrane hydrodynamic model allows for the simultaneous determination of the intermonolayer friction coefficient and the substrate-membrane friction coefficient, without any prior assumptions on the strengths of the relevant interactions. Remarkably, our calculations suggest that the viscosity of the interfacial water confined between the membrane and the substrate is elevated by ∼104 as compared to bulk water. Using hidden Markov model analysis, we then obtain insight into the transbilayer movement of lipids. We find that lipid flip-flop dynamics are very fast, with half times in the range of seconds. Importantly, we find little evidence for membrane defect mediated lipid flip-flop for SLBs at temperatures well above the solid-to-liquid transition, though defects seem to be involved when the SLBs are cooled down. Our work thus shows that the combination of single particle tracking and advanced hydrodynamic modeling provides a powerful means to obtain insight into membrane dynamics.

Polymer brushes in solid-state nanopores form an impenetrable entropic barrier for proteins.

Polymer brushes are widely used to prevent the adsorption of proteins, but the mechanisms by which they operate have remained heavily debated for many decades. We show conclusive evidence that a polymer brush can be a remarkably strong kinetic barrier towards proteins by using poly(ethylene glycol) grafted to the sidewalls of pores in 30 nm thin gold films. Despite consisting of about 90% water, the free coils seal apertures up to 100 nm entirely with respect to serum protein translocation, as monitored label-free through the plasmonic activity of the nanopores. The conclusions are further supported by atomic force microscopy and fluorescence microscopy. A theoretical model indicates that the brush undergoes a morphology transition to a sealing state when the ratio between the extension and the radius of curvature is approximately 0.8. The brush-sealed pores represent a new type of ultrathin filter with potential applications in bioanalytical systems.

Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex.

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinß1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

Strongly stretched protein resistant poly(ethylene glycol) brushes prepared by grafting-to.

We present a new grafting-to method for resistant "non-fouling" poly(ethylene glycol) brushes, which is based on grafting of polymers with reactive end groups in 0.9 M Na2SO4 at room temperature. The grafting process, the resulting brushes, and the resistance toward biomolecular adsorption are investigated by surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy. We determine both grafting density and thickness independently and use narrow molecular weight distributions which result in well-defined brushes. High density (e.g., 0.4 coils per nm(2) for 10 kDa) and thick (40 nm for 20 kDa) brushes are readily achieved that suppress adsorption from complete serum (10× dilution, exposure for 50 min) by up to 99% on gold (down to 4 ng/cm(2) protein coverage). The brushes outperform oligo(ethylene glycol) monolayers prepared on the same surfaces and analyzed in the same manner. The brush heights are in agreement with calculations based on a simple model similar to the de Gennes "strongly stretched" brush, where the height is proportional to molecular weight. This result has so far generally been considered to be possible only for brushes prepared by grafting-from. Our results are consistent with the theory that the brushes act as kinetic barriers rather than efficient prevention of adsorption at equilibrium. We suggest that the free energy barrier for passing the brush depends on both monomer concentration and thickness. The extraordinary simplicity of the method and good inert properties of the brushes should make our results widely applicable in biointerface science.

Karyopherin-centric control of nuclear pores based on molecular occupancy and kinetic analysis of multivalent binding with FG nucleoporins.

Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinß1 (Kapß1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapß1 molecules at physiological Kapß1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapß1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapß1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex.

Non-interacting molecules as innate structural probes in surface plasmon resonance.

Determining the structural parameters of a molecular layer remains an unresolved problem in surface plasmon resonance (SPR). Given that molecular form and function are intimately coupled, a breakthrough in this area could be of considerable benefit to the study of protein and/or polymer-decorated material interfaces that are ubiquitous in biology and technology. Here, we describe how noninteracting molecules function as innate structural probes that "feel" the intrinsic exclusion volume of a surface-tethered molecular layer in SPR. Importantly, this is noninvasive and provides a means to bypass the refractive index (RI) constraint that convolutes and hinders SPR thickness measurements. To show proof-of-concept, we use BSA molecules in solution to measure the thicknesses of polyethylene glycol (PEG) molecular brushes as a function of molecular weight. The SPR-acquired brush thicknesses scale with PEG hydrodynamic diameter and are in good agreement with atomic force microscopy force-distance measurements. Theoretical treatments that account for changes in the evanescent field decay length at the metal-dielectric interface indicate that the method is most appropriate for low RI layers with an estimated maximal error of ±15% in the thickness due to the RI constraint. Such in situ thickness measurements can be easily incorporated into routine SPR binding assays for investigating mesoscopic structure-function correlations of diverse molecular layers (i.e., biointerfaces).

Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes.

Conformational changes at supramolecular interfaces are fundamentally coupled to binding activity, yet it remains a challenge to probe this relationship directly. Within the nuclear pore complex, this underlies how transport receptors known as karyopherins proceed through a tethered layer of intrinsically disordered nucleoporin domains containing Phe-Gly (FG)-rich repeats (FG domains) that otherwise hinder passive transport. Here, we use nonspecific proteins (i.e., BSA) as innate molecular probes to explore FG domain conformational changes by surface plasmon resonance. This mathematically diminishes the surface plasmon resonance refractive index constraint, thereby providing the means to acquire and correlate height changes in a surface-tethered FG domain layer to Kap binding affinities in situ with respect to their relative spatial arrangements. Stepwise measurements show that FG domain collapse is caused by karyopherin ß1 (Kapß1) binding at low concentrations, but this gradually transitions into a reextension at higher Kapß1 concentrations. This ability to self-heal is intimately coupled to Kapß1-FG binding avidity that promotes the maximal incorporation of Kapß1 into the FG domain layer. Further increasing Kapß1 to physiological concentrations leads to a "pileup" of Kapß1 molecules that bind weakly to unoccupied FG repeats at the top of the layer. Therefore, binding avidity does not hinder fast transport per se. Revealing the biophysical basis underlying the form-function relationship of Kapß1-FG domain behavior results in a convergent picture in which transport and mechanistic aspects of nuclear pore complex functionality are reconciled.