Mechanism of Reversible Peptide–Bilayer Attachment: Combined Simulation and Experimental Single-Molecule Study

The binding of peptides and proteins to lipid membrane surfaces is of fundamental importance for many membrane-mediated cellular processes. Using closely matched molecular dynamics simulations and atomic force microscopy experiments, we study the force-induced desorption of single peptide chains from phospholipid bilayers to gain microscopic insight into the mechanism of reversible attachment. This approach allows quantification of desorption forces and decomposition of peptide–membrane interactions into energetic and entropic contributions. In both simulations and experiments, the desorption forces of peptides with charged and polar side chains are much smaller than those for hydrophobic peptides. The adsorption of charged/polar peptides to the membrane surface is disfavored by the energetic components, requires breaking of hydrogen bonds involving the peptides, and is favored only slightly by entropy. By contrast, the stronger adsorption of hydrophobic peptides is favored both by energy and by entropy and the desorption forces increase with increasing side-chain hydrophobicity. Interestingly, the calculated net adsorption free energies per residue correlate with experimental results of single residues, indicating that side-chain free energy contributions are largely additive. This observation can help in the design of peptides with tailored adsorption properties and in the estimation of membrane binding properties of peripheral membrane proteins

Peptide Desorption Kinetics from Single Molecule Force Spectroscopy Studies

We use a combined experimental/theoretical approach to determine the intrinsic monomeric desorption rate <i>k</i>₀ of polytyrosine and polylysine homopeptides from flat surfaces. To this end, single polypeptide molecules are covalently attached to an AFM cantilever tip and desorbed from hydrophobic self-assembled monolayers in two complementary experimental protocols. In the constant-pulling-velocity protocol, the cantilever is moved at finite velocity away from the surface and the distance at which the constant plateau force regime ends and the polymer detaches is recorded. In the waiting-time protocol, the cantilever is held at a fixed distance above the surface and the time until the polymer detaches is recorded. The desorption plateau force is varied between 10 and 90 pN, by systematically changing the aqueous solvent quality via the addition of ethanol or salt. A simultaneous fit of the experimental data from both protocols with simple two-state kinetic polymer theory allows to unambiguously disentangle and determine the model parameters corresponding to polymer contour length <i>L</i>, Kuhn length <i>a</i>, adsorption free energy λ, and intrinsic monomeric desorption rate <i>k</i>₀. Crucial to our analysis is that a statistically significant number of single-polymer desorption experiments are done with one and the same single polymer molecule for different solvent qualities. The surprisingly low value of about <i>k</i>₀ ≈ 10⁵ Hz points to significant cooperativity in the desorption process of single polymers

Highly Hydrated Deformable Polyethylene Glycol-Tethered Lipid Bilayers

The realization of a solid-supported lipid bilayer acting as a workbench for the study of membrane processes is a difficult task. For robustness, the bilayer has to be tethered to the substrate. At the same time, diffusion of the lipids and plastic deformations of the membrane should not be obstructed. Furthermore, a highly hydrated surrounding is mandatory. Here, we show that grafting of a polyethylene glycol–lipid construct (PEG2000–DSPE) to a silicon oxide surface via multiple-step silane chemistry and subsequent deposition of lipids by spin-coating result in a cushioned membrane that has the desired properties. Neutron and X-ray reflectometry measurements are combined to access thickness, density, and hydration of the bilayer and the PEG cushion. We observe a spacer of 55 Å thickness between lipid bilayer and silicon-oxide surface with a rather high hydration of up to 90 ± 3% water. While 11.5 ± 3% of the lipids are grafted to the surface, as determined from the neutron data, the diffusion constant of the lipids, as probed by diffusion of 0.5% Texas Red labeled lipids, remains rather large (<i>D</i> = 2.1 ± 0.1 μm²/s), which is a reduction of only 12% compared to a supported lipid bilayer reference without immobilized lipids. Finally, AFM indentation confirms the plastic behavior of the membrane against deformation. We show that rupture of the bilayer does not occur before the deformation exceeds 40 Å. Altogether, the presented PEG-tethered lipid bilayer mimics the deformability of natural cell membranes much better than standard solid-supported lipid bilayers

Stick-Slip Mechanisms at the Nanoscale

<div><p>When two surfaces slide past each other, energy is mainly dissipated by stick-slip events. Macroscopic stick-slip is usually explained by asperities that come in and out of contact. Herein, we probe stick-slip at the nanoscale at interfaces and polymer coated interfaces by pulling single polymers covalently attached to an AFM cantilever tip laterally over solid substrates in liquid environment. We find two different stick mechanisms, namely desorption stick (DS) and cooperative stick (CS). While DS-slip resembles the velocity dependence of macroscopic stick-slip, CS-slip shows an increase in friction with velocity. For various reasons we anticipate that both stick mechanisms are necessary for a molecular understanding of stick-slip at the interface and interphase. </p></div

On the Relationship between Peptide Adsorption Resistance and Surface Contact Angle: A Combined Experimental and Simulation Single-Molecule Study

The force-induced desorption of single peptide chains from mixed OH/CH₃-terminated self-assembled monolayers is studied in closely matched molecular dynamics simulations and atomic force microscopy experiments with the goal to gain microscopic understanding of the transition between peptide adsorption and adsorption resistance as the surface contact angle is varied. In both simulations and experiments, the surfaces become adsorption resistant against hydrophilic as well as hydrophobic peptides when their contact angle decreases below θ ≈ 50°–60°, thus confirming the so-called Berg limit established in the context of protein and cell adsorption. Entropy/enthalpy decomposition of the simulation results reveals that the key discriminator between the adsorption of different residues on a hydrophobic monolayer is of entropic nature and thus is suggested to be linked to the hydrophobic effect. By pushing a polyalanine peptide onto a polar surface, simulations reveal that the peptide adsorption resistance is caused by the strongly bound water hydration layer and characterized by the simultaneous gain of both total entropy in the system and total number of hydrogen bonds between water, peptide, and surface. This mechanistic insight into peptide adsorption resistance might help to refine design principles for anti-fouling surfaces

Cohesion Mechanisms of Polystyrene-Based Thin Polymer Films

The cohesion mechanisms of end-functionalized high molar mass polystyrene with very low polydispersity (PS, <i>M</i>_n = 1.26 × 10⁶ g mol^–1, PDI = 1.06) and polylysine (PLL, 150–130 × 10³ g mol^–1) on silicon (Si) supported thin PS films are investigated by desorbing single polymers covalently bound to an atomic force microscope (AFM) cantilever tip. The influence of film preparation conditions and film architecture on polymer cohesion mechanisms is probed by comparing spin-coated PS films (scPS) with a thickness range of 6–52 nm and covalently surface-attached PS films (saPS) with a thickness of 15–83 nm. Annealed scPS prevents cohesion of further PS polymers unless the scPS partly dewets. In all other cases, two different cohesion mechanisms are observed: first, a previously described equilibrium desorption similar to hydrophobic solid substrate desorption, represented by a plateau of constant force in the force–extension curve, and second, a nonequilibrium mechanism with nonlinear force–extension behavior. The second requires a geometrical interlock between the tip bound single molecule and the PS film. Remarkably, this mechanism is observed below the glass transition temperature of PS films and is promoted by good solvent conditions. These findings contrast many bulk measurements assuming a glassy state of the complete polymer film, but they are consistent with fluid like boundary layers having a high mobility. Our results further underline the decisive influence of polymer film conformation and mobility close to its solvent exposed boundary layer for the cohesion of polymer coatings

Effect of Molecular Architecture on Single Polymer Adhesion

Several applications require strong noncovalent adhesion of polymers to substrates. Graft and branched polymers have proven superior to linear polymers, but the molecular mechanism is still unclear. Here, this question is addressed on the single molecule level with an atomic force microscopy (AFM) based method. It is determined how the presence of side chains and their molecular architecture influence the adhesion and the mobility of polymers on solid substrates. Surprisingly, the adhesion of mobile polymers cannot significantly be improved by side chains or their architecture. Only for immobile polymers a significantly higher maximum rupture force for graft, bottle-brush, and branched polymers compared to linear chains is measured. Our results suggest that a combination of polymer architecture and strong molecular bonds is necessary to increase the polymer–surface contact area. An increased contact area together with intrachain cohesion (e.g., by entanglements) leads to improved polymer adhesion. These findings may prove useful for the design of stable polymer coatings

Dye Labeling and DNA Packaging of φ29 Proheads

<div><p>(A) SDS-PAGE of 170C-connector-mutant proheads. Protein stain of proheads shows the structural components gp8 (capsid), gp8.5 (fiber), gp10 (connector), and gp7 (scaffold) in lane (a). Fluorescence scan of the gel showing labeled proheads with various amounts of dye per gp10 monomer used in labeling reaction: 1 dye per gp10, lane (b); 0.5 dyes per gp10, lane (c); 0.25 dyes per gp10, lane (d); 0.125 dyes per gp10, lane (e); 0.0625 dyes per gp10, lane (f); and no dye, lane (g). The bands in the fluorescence scan with no match in the protein stain originate from highly reactive but quantitatively minor E. coli proteins.</p> <p>(B) DNA packaging tested by nuclease (EcoRI) protection assay using the labeled proheads from (A). Lane (a) shows input DNA-gp3; lane (b) shows a negative (no ATP) control. Packaged DNA is protected from nuclease digestion. Packaging activity is unaffected by dye labeling when compared to a 193C packaging control, lane (c). (B) Shows labeled proheads from (A), ranging from 1 dye per gp10, lane (d); 0.5 dyes per gp10, lane (e); through to no dye, lane (i).</p></div

Fluorescence Polarization Studies of Dye-Labeled, Stalled-Packaging Complexes

<p>Dye-labeled, stalled-packaging complexes were attached to the surface of a flow chamber and excited using the total internal reflection microscope. The excitation polarization was rotated between s- and p-polarization with a frequency of 0.7 Hz. The emitted fluorescence was separated into s- and p-polarization, respectively, and simultaneously detected (black and red). The dye bleached after 22 s. The integration time per data point was 75 ms. a.u., arbitrary units.</p

Fluorescence Signal from Packaging Complexes

<p>The graphs show the fluorescence intensity of vertical (black) and horizontal (red) polarization detected simultaneously. One example for each of the six investigated mutants is shown. The data were recorded with an integration time of 75 ms and three-points smoothing was applied. The camera background (closed shutter) was subtracted. The signal was normalized using the fluorescence intensity of the bead after packaging was completed. The traces are vertically shifted for clarity. The inset shows a zoom of the data. Here, the unfiltered data is displayed (scatter) together with the three-point sliding average. (A) Fluorescence recorded for a single complex of mutant 170C. The shutter is opened after about half a second. After t = 4 s the first dye bleaches and after 19 s the second. After about t = 100 s the fluorescence signal again starts to increase, since the magnetic bead is pulled into the evanescent field as the prohead reels in the DNA. The signal increases in an exponential fashion as the fluorescent bead samples the intensity profile of the evanescent wave. Similar behavior was observed for (B) 97C; (C) 168C; (D) 189C; (E) 190C; and (F) 260C.</p