Computational reconstruction of multidomain proteins using atomic force microscopy data

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.     

Single and multiple bonds in (strept)avidin-biotin interactions

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures (?<1 ?), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.     

Atomic force microscope,molecular imaging,and analysis

Image visibility is a central issue in analyzing all kinds of microscopic images. An increase of intensity contrast helps to raise the image visibility, thereby to reveal fine image features. Accordingly, a proper evaluation of results with current imaging parameters can be used for feedback on future imaging experiments. In this work, we have applied the Laplacian function of image intensity as either an additive component (Laplacian mask) or a multiplying factor (Laplacian weight) for enhancing image contrast of high‐resolution AFM images of two molecular systems, an unknown protein imaged in air, provided by AFM COST Action TD1002 ( http://www.afm4nanomedbio.eu /), and tobacco mosaic virus (TMV) particles imaged in liquid. Based on both visual inspection and quantitative representation of contrast measurements, we found that the Laplacian weight is more effective than the Laplacian mask for the unknown protein, whereas for the TMV system the strengthened Laplacian mask is superior to the Laplacian weight. The present results indicate that a mathematical function, as exemplified by the Laplacian function, may yield varied processing effects with different operations. To interpret the diversity of molecular structure and topology in images, an explicit expression for processing procedures should be included in scientific reports alongside instrumental setups. Copyright © 2015 John Wiley & Sons, Ltd.     

Can semiochemicals attract both western flower thrips and their anthocorid predators?

Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) (western flower thrips, WFT) is a key pest in a range of crops worldwide. Anthocorid species (Hemiptera) are important natural enemies of thrips. Several experiments were undertaken to determine whether a thrips lure, methyl isonicotinate (MI), affected the behaviour of WFT and anthocorids found in outdoor crops. Currently, this volatile compound is used in conjunction with sticky traps for monitoring thrips predominantly in greenhouses in Northern Europe. In the present study, in a nectarine [Prunus persica (L.) (Rosaceae)] orchard and an outdoor capsicum [Capsicum annuum L. (Solanaceae)] crop in Spain, blue sticky and white water traps with MI caught significantly more WFT than traps without MI. The volatile compound also significantly increased both blue sticky and white water trap capture of anthocorids – predominantly Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) – in the capsicum crop. These results indicate that the behaviour of both WFT and O. laevigatus were altered by the presence of the compound and suggest there is potential to develop novel tools based on MI in conjunction with biological control strategies for thrips management.     

Visually and olfactorily enhanced attractive devices for thrips management

‘Lure-and-infect’ is an insect pest management strategy with high potential but so far there are few examples of its application. Using traps as surrogates for auto-dissemination devices, we tested the attractiveness to naturally occurring thrips (Thysanoptera: Thripidae) of three trap types differing in colour and structure, with and without the thrips lure methyl isonicotinate (MI), and sticky plate traps as a control. The aim was to find more effective traps that could be further developed into devices for auto-dissemination and lure-and-infect of thrips. The number of thrips captured varied substantially with trap type and the presence of the MI lure. We found a high visual response to a sticky ‘white ruffle’ trap (i.e., a 30-cm-long cylindrical outline of folded fabric), compared to a commonly used blue sticky plate trap (Bug-scan) as the control. This effect was seen both in a greenhouse with roses (Rosa spp.), where we encountered western flower thrips, Frankliniella occidentalis (Pergande), and in a grass field, where we encountered onion thrips, Thrips tabaci Lindeman, and New Zealand flower thrips, Thrips obscuratus (Crawford). In the absence of MI, the white ruffle trap caught 7–22× more thrips than the control Bug-scan trap. A similarly designed blue ruffle trap and a modified Lynfield trap caught lower thrips numbers than the white ruffle and the control Bug-scan traps. Presence of MI substantially increased the captures of T. tabaci in all three trap types in the field (2.5–18×). In the greenhouse, without MI the white ruffle trap caught 3.5–14× more thrips than the Bug-scan, blue ruffle, or modified Lynfield traps. Presence of MI increased the captures of F. occidentalis males and females in the Lynfield and blue ruffle traps (1.4–2.8×), but not in the white ruffle trap in the greenhouse (ca. 1.1×). The importance of visual and olfactory factors for the design of effective auto-dissemination and lure-and-infect strategies for thrips management is discussed.     

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.     

A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-) > Br(-)--NO3(-) > F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.     

The discovery of a new species of Ceranisus from southwestern Turkey

An explorative trip was made to southwesternTurkey in April 1995 in search of pear thrips,Taeniothrips inconsequens (Uzel)(Thysanoptera: Thripidae), and its naturalenemies. Forty-three females and 13 males of apreviously undescribed species of the genusCeranisus Walker (Hymenoptera:Eulophidae) were found, mainly in floweringArbutus andrachne L. Although theparasitoid was found in association withseveral thrips species, but especially T.inconsequens adults and larvae, its host orhosts were not determined. The parasitoid isdescribed and given the name Ceranisusantalyacus S. Triapitsyn.