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Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. This pitfall may be alleviated by torsional constraints. Confidence in detected transitions may be increased by using conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution.

The solution processability of organic semiconductors and conjugated polymers along with the advent of nanomaterials as conducting inks have revolutionized next-generation flexible consumer electronics. Another equally important class of nanomaterials, self-assembled peptides, heralded as next-generation materials for bioelectronics, have a lot of potential in printed technology. In this minireview, we address the self-assembly process in dipeptides, their application in electronics, and recent progress in three-dimensional printing. The prospect of a generalizable path for nanopatterning self-assembled peptides using ice lithography and its challenges are further discussed.

Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores by atomic force microscopy revealed co-existence of simple depressions and more complex pores, which are likely formed by CL assembled in an alternate oligomer orientation. We propose that this structural rearrangement represents a maturation mechanism that stabilizes pore formation to achieve more robust cellular damage. To summarize, CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

Cortical bone porosity is intimately linked with remodeling, is of growing clinical interest, and is increasingly accessible by imaging. Thus, the potential of animal models of osteoporosis (OP) to provide a platform for studying how porosity develops and responds to interventions is tremendous. To date, rabbit models of OP have largely focused on trabecular microarchitecture or bone density; some such as ovariectomy (OVX) have uncertain efficacy and cortical porosity has not been extensively reported. Our primary objective was to characterize tibial cortical porosity in rabbit‐based models of OP, including OVX, glucocorticoids (GC), and OVX + GC relative to controls (SHAM). We sought to: (i) test the hypothesis that intracortical remodeling is elevated in these models; (ii) contrast cortical remodeling and porosity in these models with that induced by parathyroid hormone (1–34; PTH); and (iii) contrast trabecular morphology in the proximal tibia across all groups. Evidence that an increase in cortical porosity occurred in all groups was observed, although this was the least robust for GC. Histomorphometric measures supported the hypothesis that remodeling rate was elevated in all groups and also revealed evidence of uncoupling of bone resorption and formation in the GC and OVX + GC groups. For trabecular bone, a pattern of loss was observed for OVX, GC, and OVX + GC groups, whereas the opposite was observed for PTH. Change in trabecular number best explained these patterns. Taken together, the findings indicated rabbit models provide a viable and varied platform for the study of OP and associated changes in cortical remodeling and porosity. Intriguingly, the evidence revealed differing effects on the cortical and trabecular envelopes for the PTH model. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bilayers. The association of SecA with SecYEG, forming membrane-bound SecYEG/SecA translocases, is directly visualized. Recent work showing topographical aspects of the translocation process that vary with precursor species is also discussed. The data suggests that the Sec system does not employ a single translocation mechanism. We posit that differences in the spatial frequency distribution of hydrophobic content within precursor sequences may be a determining factor in mechanism selection. Precise AFM investigations of active translocases are poised to advance our currently vague understanding of the complicated macromolecular movements underlying protein export across membranes.

The fungus

Surface-supported lipid bilayers are used widely throughout the nanoscience community as cellular membrane mimics. For example, they are frequently employed in single-molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding. However, in AFM as well as in other surface-sensing techniques, the close proximity of the supporting surface raises questions about preservation of the biochemical activity. Employing the model translocase from the general secretory (Sec) system of Escherichia coli, here we quantify the activity via two biochemical assays in surface-supported bilayers. The first assesses ATP hydrolysis and the second assesses polypeptide translocation across the membrane via protection from added protease. Hydrolysis assays revealed distinct levels of activation ranging from medium (translocase-activated) to high (translocation-associated) that were similar to traditional solution experiments and further identified an adenosine triphosphatase population exhibiting characteristics of conformational hysteresis. Translocation assays revealed turn over numbers that were comparable to solution but with a 10-fold reduction in apparent rate constant. Despite differences in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topographic complexity of the underlying surface. Rough glass coverslips were favored over atomically flat mica, likely due to differences in frictional coupling between the translocating polypeptide and surface. Neutron reflectometry and AFM corroborated the biochemical measurements and provided structural characterization of the submembrane space and upper surface of the bilayer. Overall, the translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.

Using synthetic molecular evolution, we previously discovered a family of peptides that cause macromolecular poration in synthetic membranes at low peptide concentration in a way that is triggered by acidic pH. To understand the mechanism of action of these "pHD peptides", here we systematically explored structure-function relationships through measurements of the effect of pH and peptide concentration on membrane binding, peptide structure, and the formation of macromolecular-sized pores in membranes. Both AFM and functional assays demonstrate the peptide-induced appearance of large pores in bilayers. Pore formation has a very steep pH dependence and is also dependent on peptide concentration. In vesicles, 50% leakage of 40 kDa dextrans occurs at 1 bound peptide per 1300 lipids or only 75 peptides per vesicle, an observation that holds true across a wide range of acidic pH values. The major role of pH is to regulate the amount of peptide bound per vesicle. The physical chemistry and sequence of the pHD peptides affect their potency and pH dependence; therefore, the sequence-structure-function relationships described here can be used for the future design and optimization of membrane permeabilizing peptides for specific applications.

Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.

Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.

A number of peptides are known to bind lipid bilayer membranes and cause these natural barriers to leak in an uncontrolled manner. Though membrane permeabilizing peptides play critical roles in cellular activity and may have promising future applications in the therapeutic arena, significant questions remain about their mechanisms of action. The atomic force microscope (AFM) is a single molecule imaging tool capable of addressing lipid bilayers in near-native fluid conditions. The apparatus complements traditional assays by providing local topographic maps of bilayer remodeling induced by membrane permeabilizing peptides. The information garnered from the AFM includes direct visualization and statistical analyses of distinct bilayer remodeling modes such as highly localized pore-like voids in the bilayer and dispersed thinned membrane regions. Colocalization of distinct remodeling modes can be studied. Here we examine recent work in the field and outline methods used to achieve precise AFM image data. Experimental challenges and common pitfalls are discussed as well as techniques for unbiased analysis including the Hessian blob detection algorithm, bootstrapping, and the Bayesian information criterion. When coupled with robust statistical analyses, high precision AFM data is poised to advance understanding of an important family of peptides that cause poration of membrane bilayers.

Protein-lipid interfaces are among the most fundamental in biology. Yet applying conventional techniques to study the biophysical attributes of these systems is challenging and has left many unknowns. For example, what is the kinetic pathway and energy landscape experienced by a polypeptide chain when in close proximity to a fluid lipid bilayer? Here we review the experimental and theoretical progress we have made in addressing this question from a single molecule perspective. Some remaining impediments are also discussed.

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.

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The K(m) of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (K(D)s) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a “closed” conformation, while DOX shifts Pgp to an “intermediate” conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed “small” and “large” protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these “small” and “large” protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 A AFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the “small” and “large” protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

Systematically evolved from the primary active component of bee venom, MelP5 is a lipophilic peptide with important physical properties that differ from wild-type melittin, including the ability to create large equilibrium pores in lipid bilayers at low peptide to lipid ratios. Self-assembly into stable membrane spanning pores makes MelP5 a promising candidate for future applications in the pharmaceutical arena. Despite significant interest, little is known about the mechanism by which MelP5 remodels the lipid bilayer upon binding. We demonstrate by direct atomic force microscope imaging of supported lipid bilayers in solution that MelP5 remodels 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) in one of two ways. It creates either highly localized voids in the bilayer or diffuse nonlocalized thinning. Thinning of the bilayer was measured to be 3.0 ± 1.4 Å (mean ± standard deviation) below the surface of the upper leaflet of the bilayer. Pores, defined as highly localized voids in the bilayer, exhibited several sizes. Approximately 20% of pores exhibited large footprint areas (47 ± 20 nm

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.

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

Based on a well-preserved specimen from Eocene Baltic amber, the second fossil species belonging to the genus Pycnomerus Erichson (Coleoptera: Zopheridae: Zopherinae), P. agtsteinicus Bukejs, Alekseev & McKellar sp. nov. is described and illustrated using synchrotron X-ray micro-CT observations. The new species adds to the sparse fossil record of Pycnomerus, which consists of the Baltic amber discoveries, and only four subfossil records in sediments that are less than one million years old. As part of this work, the new replacement name Pycnomerus lordi Bukejs, Alekseev & McKellar nom. nov. is also established for the extant species Pycnomerus sulcicollis LeConte, 1863 [non Pycnomerus sulcicollis (Germar, 1824)].

We reveal the dynamic topography of active translocases as a function of precursor, nucleotide, and stage of translocation., Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

Background: Residential woodsmoke is an under-regulated source of fine particulate matter (PM2.5), often surpassing mobile and industrial emissions in rural communities in North America and elsewhe...

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.

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning α-helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

Background/Aim: In recent years, many air quality monitoring programs have favoured measurement of particles less than 2.5 microns (PM2.5) over particles less than 10 microns (PM10) in light of evi...

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 (

The selective hydrogenation of furfuryl alcohol was investigated at room temperature by using supported palladium catalysts. The catalysts are very selective to the formation of 2-methylfuran. Furthermore, the addition of tin to palladium showed similar catalytic activity, but was more selective to tetrahydrofurfuryl alcohol. Variation of the Sn/Pd ratio has shown a considerable and interesting effect on the selectivity pattern. Addition of a small amount of Sn (1 wt %) shifted the selectivity towards tetrahydrofurfuryl alcohol and methyltetrahydrofuran, which are ring-saturated molecules. Increasing the tin ratio further decreased the catalytic activity and also showed very poor selectivity to either of these products.

Hemicoelus favonii sp. nov. is described and illustrated from Eocene Baltic amber. This new fossil species differs from extant congeners in having 11-segmented antennae; a metathoracic ventrite with large impression in its anterior portion; a pronotum distinctly narrower than the elytral base region; the posterior suture of abdominal ventrite 1 weakly arcuate medially; sharp lateral pronotal margins that are incomplete and distinct in their basal half only; elytral striae that are not grouped in pairs; posterior pronotal angles that are rounded; elytral intestriae 3, 5, 7 and 9 that are distinctly convex; and a comparatively small total body size. The presence of Hemicoelus in Baltic amber suggests that moist, rotting wood was available as a microhabitat in the ancient forest. Beyond the new species description, the systematic placement of Anobium jacquelinae Hawkeswood, Makhan & Turner is discussed. A new fossil record for Microbregma waldwico Bukejs & Alekseev, and the first report of the genus Trichodesma LeConte from Eocene Baltic amber are also presented.

In recent years, many air quality monitoring programs have favored measurement of particles less than 2.5 µm (PM2.5) over particles less than 10 µm (PM10) in light of evidence that health impacts are mostly from the fine fraction. However, the coarse fraction (PM10-2.5) may have independent health impacts that support continued measurement of PM10 in some areas, such as those affected by road dust. The objective of this study was to evaluate the associations between different measures of daily PM exposure and two daily indicators of population health in seven communities in British Columbia, Canada, where road dust is an ongoing concern. The measures of exposure were PM10, PM2.5, PM10-2.5, PM2.5 adjusted for PM10-2.5, and PM10-2.5 adjusted for PM2.5. The indicators of population health were dispensations of the respiratory reliever medication salbutamol sulfate and nonaccidental mortality. This study followed a time-series design using Poisson regression over a 2003–2015 study period, with analyse...

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.

The selective hydrogenation of furfuryl alcohol into 2-methylfuran was investigated at room temperature using palladium supported catalysts. We have shown that Pd–TiO2 catalysts can be very effective for the synthesis of 2-methylfuran at room temperature and low pressure of hydrogen (1–3 bar). The effect of various reaction conditions (pressure, catalyst amount, and solvent) was studied.

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.

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.

Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude (∼1–10 A) and were attributed to the two large loops of SecY linking transmembrane helices 6–7 and 8–9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.

Residential woodsmoke is an under-regulated source of fine particulate matter (PM2.5), often surpassing mobile and industrial emissions in rural communities in North America and elsewhere. In the province of British Columbia (BC), Canada, many municipalities are hesitant to adopt stricter regulations for residential wood burning without empirical evidence that smoke is affecting local air quality. The objective of this study was to develop a retrospective algorithm that uses 1-h PM2.5 concentrations and daily temperature data to identify smoky days in order to prioritise communities by smoke impacts. Levoglucosan measurements from one of the smokiest communities were used to establish the most informative values for three algorithmic parameters: the daily standard deviation of 1-h PM2.5 measurements; the daily mean temperature; and the daytime-to-nighttime ratio of PM2.5 concentrations. Alternate parameterizations were tested in 45 sensitivity analyses. Using the most informative parameter values on the most recent two years of data for each community, the number of smoky days ranged from 5 to 277. Heat maps visualizing seasonal and diurnal variation in PM2.5 concentrations showed clear differences between the higher- and lower-ranked communities. Some communities were sensitive to one or more of the parameters, but the overall rankings were consistent across the 45 analyses. This information will allow stakeholder agencies to work with local governments on implementing appropriate intervention strategies for the most smoke-impacted communities.