Effect of CM15 on Supported Lipid Bilayer Probed by Atomic Force Microscopy.

Antimicrobial peptides are key components of the immune system. These peptides affect the membrane in various ways; some form nano-sized pores, while others only produce minor defects. Since these peptides are increasingly important in developing antimicrobial drugs, understanding the mechanism of their interactions with lipid bilayers is critical. Here, using atomic force microscopy (AFM), we investigated the effect of a synthetic hybrid peptide, CM15, on the membrane surface comprising E. coli polar lipid extract. Direct imaging of supported lipid bilayers exposed to various concentrations of the peptide revealed significant membrane remodeling. We found that CM15 interacts with supported lipid bilayers and forms membrane-spanning defects very quickly. It is found that CM15 is capable of remodeling both leaflets of the bilayer. For lower CM15 concentrations, punctate void-like defects were observed, some of which re-sealed themselves as a function of time. However, for CM15 concentrations higher than 5 µM, the defects on the bilayers became so widespread that they disrupted the membrane integrity completely. This work enhances the understanding of CM15 interactions with the bacterial lipid bilayer.

Characterizing the Locus of a Peripheral Membrane Protein-Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli.

Quantitative characterization of the strength of peripheral membrane protein-lipid bilayer interactions is fundamental in the understanding of many protein targeting pathways. SecA is a peripheral membrane protein that plays a central role in translocating precursor proteins across the inner membrane of E. coli. The membrane binding activity of the extreme N-terminus of SecA is critical for translocase function. Yet, the mechanical strength of the interaction and the kinetic pathways that this segment of SecA experiences when in proximity of an E. coli polar lipid bilayer has not been characterized. We directly measured the N-terminal SecA-lipid bilayer interaction using precision single molecule atomic force microscope (AFM)-based dynamic force spectroscopy. To provide conformational data inaccessible to AFM, we also performed all-atom molecular dynamics simulations and circular dichroism measurements. The N-terminal 10 amino acids of SecA have little secondary structure when bound to zwitterionic lipid head groups, but secondary structure, which rigidifies the lipid-bound protein segment, emerges when negatively charged lipids are present. Analysis of the single molecule protein-lipid dissociation data converged to a well-defined lipid-bound-state lifetime in the absence of force, τ0lipid = 0.9 s, which is well separated from and longer than the fundamental time scale of the secretion process, defined as the time required to translocate a single amino acid residue (∼50 ms). This value of τ0lipid is likely to represent a lower limit of the in vivo membrane-bound lifetime due to factors including the minimal system employed here.

Multiple stochastic pathways in forced peptide-lipid membrane detachment.

We have used high resolution AFM based dynamic force spectroscopy to investigate peptide-lipid membrane interactions by measuring the detachment (last-rupture) force distribution, P(F), and the corresponding force dependent rupture rate, k(F), for two different peptides and lipid bilayers. The measured quantities, which differed considerably for different peptides, lipid-membranes, AFM tips (prepared under identical conditions), and retraction speeds of the AFM cantilever, could not be described in terms of the standard theory, according to which detachment occurs along a single pathway, corresponding to a diffusive escape process across a free energy barrier. In particular, the prominent retraction speed dependence of k(F) was a clear indication that peptide-lipid membrane dissociation occurs stochastically along several detachment pathways. Thereby, we have formulated a general theoretical approach for describing P(F) and k(F), by assuming that peptide detachment from lipid membranes occurs, with certain probability, along a few dominant diffusive pathways. This new method was validated through a consistent interpretation of the experimental data. Furthermore, we have found that for moderate retraction speeds at intermediate force values, k(F) exhibits catch-bond behavior (i.e. decreasing detachment rate with increasing force). According to the proposed model this behavior is due to the stochastic mixing of individual detachment pathways which do not convert or cross during rupture. To our knowledge, such catch-bond mechanism has not been proposed and demonstrated before for a peptide-lipid interaction.

High-Resolution AFM-Based Force Spectroscopy.

Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen significant enhancements in both force and time resolution in recent years. This chapter details two AFM cantilever modification procedures that yield high force precision over different temporal bandwidths. Specifically, it explains a fairly straightforward method to achieve sub-pN force precision and stability at low frequencies (<50 Hz) by removing the metal coatings from a commercially available cantilever. A more involved procedure utilizing a focused ion beam milling machine is required to maintain high force precision at enhanced bandwidths. Both modification methods allow site-specific attachment of biomolecules onto the apex area of the tips for force spectroscopy. The chapter concludes with a comparative demonstration using the two cantilever modification methods to study a lipid-protein interaction.

The Hessian Blob Algorithm: Precise Particle Detection in Atomic Force Microscopy Imagery.

Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias. Here, we introduce the Hessian blob to address these shortcomings. Combining a scale-space framework with measures of local image curvature, the Hessian blob formally defines particle centers and their boundaries, both to subpixel precision. Resulting particle boundaries are independent of user defined parameters, with no image preprocessing required. We demonstrate through direct comparison that the Hessian blob algorithm more accurately detects biomolecules than conventional AFM particle detection techniques. Furthermore, the algorithm proves largely insensitive to common imaging artifacts and noise, delivering a stable framework for particle analysis in AFM.

Single-Molecule Peptide-Lipid Affinity Assay Reveals Interplay between Solution Structure and Partitioning.

Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, the lack of a suitable methodology for directly probing these interactions has hindered the mechanistic understanding. We developed a precision atomic force microscopy-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) that are lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series and two copies SecA2-11 linked in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with the measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

Calorimetric study of phase transitions in ocylcyanobiphenyl-barium titanate nanoparticle dispersions.

High-resolution ac-calorimetry is reported on the weakly first-order isotropic to nematic (I-N) and the continuous nematic to smectic-A (N-SmA) phase transitions in the liquid crystal octylcyanobiphenyl (8CB) doped with a ferroelectric nanoparticle barium titanate, BaTiO3 (BT). Measurements were performed as a function of BT concentration and over a wide temperature range well above and below the two transitions. From the thermal scans of all samples (having BT mass fraction φ(m) = 0.001 to 0.014 and pure 8CB), both the I-N and the N-SmA transitions evolve in character. Specifically, there appears an unusual change of the I-N specific heat peak shape on heating as φ(m) increases. Both the transitions shift to lower temperature at a different rate for φ(m)<φ(m)(c)=0.002 as compared to that for φ(m)>φ(m)(c). The effective transition enthalpies are essentially constant and similar to that seen in the bulk. Using a simple geometric model, the mean distance between the BT particles at the cross-over φ(m)(c) is found to be x(c)~3 µm, which is consistent with an estimated surface extrapolation length b for the nematic director. This suggests that the low φ(m) regime is dominated by an impurity/disorder effect while for φ(m)>φ(m)(c) the mean distance is small enough for the LC to mediate coupling between the BT ferroelectric nanoparticles.

Three-dimensional atomic force microscopy: interaction force vector by direct observation of tip trajectory.

The prospect of a robust three-dimensional atomic force microscope (AFM) holds significant promise in nanoscience. Yet, in conventional AFM, the tip-sample interaction force vector is not directly accessible. We scatter a focused laser directly off an AFM tip apex to rapidly and precisely measure the tapping tip trajectory in three-dimensional space. This data also yields three-dimensional cantilever spring constants, effective masses, and hence, the tip-sample interaction force components via Newton's second law. Significant lateral forces representing 49 and 13% of the normal force (Fz = 152 ± 17 pN) were observed in common tapping mode conditions as a silicon tip intermittently contacted a glass substrate in aqueous solution; as a consequence, the direction of the force vector tilted considerably more than expected. When addressing the surface of a lipid bilayer, the behavior of the force components differed significantly from that observed on glass. This is attributed to the lateral mobility of the lipid membrane coupled with its elastic properties. Direct access to interaction components Fx, Fy, and Fz provides a more complete view of tip dynamics that underlie force microscope operation and can form the foundation of a three-dimensional AFM in a plurality of conditions.

Study of the isotropic to smectic-A phase transition in liquid crystal and acetone binary mixtures.

The first-order transition from the isotropic (I) to smectic-A (Sm A) phase in the liquid crystal 4-cyano-4(')-decylbiphenyl (10CB) doped with the polar solvent acetone (ace) has been studied as a function of solvent concentration by high-resolution ac-calorimetry. Heating and cooling scans were performed for miscible 10CB+ace samples having acetone mole fractions from x(ace)=0.05 (1 wt %) to 0.36 (10%) over a wide temperature range from 310 to 327 K. Two distinct first-order phase transition features are observed in the mixture whereas there is only one transition (I-Sm A) in the pure 10CB for that particular temperature range. Both calorimetric features reproduce on repeated heating and cooling scans and evolve with increasing x(ace) with the high-temperature feature relatively stable in temperature but reduced in size while the low-temperature feature shifts dramatically to lower temperature and exhibits increased dispersion. The coexistence region increases for the low-temperature feature but remains fairly constant for the high-temperature feature as a function of x(ace). Polarizing optical microscopy supports the identification of a smectic phase below the high-temperature heat capacity signature indicating that the low-temperature feature represents an injected smectic-smectic phase transition. These effects may be the consequence of screening the intermolecular potential of the liquid crystals by the solvent that stabilizes a weak smectic phase intermediate of the isotropic and pure smectic-A.

Calorimetric study of the nematic to smectic-A phase transition in octylcyanobiphenyl-hexane binary mixtures.

The continuous nematic to smectic-A (N-SmA) phase transition has been studied by high-resolution ac-calorimetry in binary mixtures of the liquid crystal octylcyanobiphenyl (8CB) and a nonmesogenic, low-molecular weight, solvent n-hexane (hex) as a function of temperature and solvent concentration. Heating and cooling scans about the N-SmA transition temperature were repeatedly performed on pure and six 8CB+hex samples having hexane molar concentration ranging from x(hex)=0.02 to 0.12. All 8CB+hex samples in this range of x(hex) remain macroscopically miscible and exhibit an N-SmA heat capacity peak that shifts nonmonotonically to lower temperature and evolves in shape, with a reproducible hysteresis, as x(hex) increases. The imaginary part of heat capacity remains zero up to x(hex)(TCP) ≃ 0.07 above which the distinct peak is observed, corresponding to a jump in both the real and imaginary enthalpy, indicating the onset of first-order behavior. A simple power-law analysis reveals an effective exponent that increases smoothly from 0.30 to 0.50 with an amplitude ratio A-/A+→1 as x(hex)→x(hex)(TCP). This observed crossover of the N-SmA toward a tricritical point driven by solvent concentration is consistent with previous results and can be understood as a solvent softening of the liquid crystal and concument promoting of nematic fluctuations.