Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

SLC5 and SLC2 transporters in epithelia-cellular role and molecular mechanisms.

Members of the SLC5 and SLC2 family are prominently involved in epithelial sugar transport. SGLT1 (sodium-glucose transporter) and SGLT2, as representatives of the former, mediate sodium-dependent uptake of sugars into intestinal and renal cells. GLUT2 (glucose transporter), as representative of the latter, facilitates the sodium-independent exit of sugars from cells. SGLT has played a major role in the formulation and experimental proof for the existence of sodium cotransport systems. Based on the sequence data and biochemical and biophysical analyses, the role of extramembranous loops in sugar and inhibitor binding can be delineated. Crystal structures and homology modeling of SGLT reveal that the sugar translocation involves operation of two hydrophobic gates and intermediate exofacial and endofacial occluded states of the carrier in an alternating access model. The same basic model is proposed for GLUT1. Studies on GLUT1 have pioneered the isolation of eukaryotic transporters by biochemical methods and the development of transport kinetics and transporter models. For GLUT1, results from extensive mutagenesis, cysteine substitution and accessibility studies can be incorporated into a homology model with a barrel-like structure in which accessibility to the extracellular and intracellular medium is altered by pinching movements of some of the helices. For SGLT1 and GLUT1, the extensive hydrophilic and hydrophobic interactions between sugars and binding sites of the various intramembrane helices occur and lead to different substrate specificities and inhibitor affinities of the two transporters. A complex network of regulatory steps adapts the transport activity to the needs of the body.

Hybrid optical-electrochemical electronic nose system based on Zn-porphyrin and multi-walled carbon nanotube composite.

In this work, we have enhanced the capability of an e-nose system based on combined optical and electrochemical transduction within a single gas sensor array. The optical part of this e-nose is based on detection of the absorption changes of light emitted from eight light emitting diodes (LEDs) as measured by a CMOS photo-detector. The electrochemical part works by measuring the change in electrical resistivity of the sensing materials upon contact with the sample vapor. Zinc-5,10,15,20-tetra-phenyl-21H,23H-porphyrin (ZnTPP) and multi-walled carbon nanotube (MWCNT) composite was used as the sensing materials based on its good optoelectronic properties. This sensing layer was characterized by UV-Vis spectroscopy and atomic force microscope and tested with various VOC vapors. Density functional theory (DFT) calculations were performed to investigate the electronic properties and interaction energies between ZnTPP and analyte molecules. It can be clearly seen that this hybrid optical-electrochemical electronic nose system can classify the vapor of different volatile organic compounds.

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells.

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.

An artificial nose based on M-porphyrin (M = Mg, Zn) thin film and optical spectroscopy.

Artificial nose has recently become an emerging instrument for quality assurance in the food industry. These paper present the optical gas sensors based on Magnesium-5, 10, 15, 20-tetra phenyl-porphyrin (MgTPP) and Zinc-5, 10, 15, 20-tetra phenyl-porphyrin (ZnTPP) thin films and their application as an artificial nose. Based on the measurement of optical absorbance response using a general UV-Vis spectroscopy, this artificial nose was tested to discriminate various volatile organic compounds (VOCs) and Thai beverages. Atomic force microscopy (AFM) and X-rays diffraction (XRD) were used to confirm the polycrystalline structure of the sensing materials. Density functional theory (DFT) calculations reveal that MgTPP interacts more strongly with the VOCs than ZnTPP, especially with methanol. The classification results of VOCs and Thai beverage vapors using the principal component analysis indicate that both MgTPP and ZnTPP-based artificial noses can be an efficient tool for quality assurance of alcoholic beverages.

A biophysical glance at the outer surface of the membrane transporter SGLT1.

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium-glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.

Three surface subdomains form the vestibule of the Na+/glucose cotransporter SGLT1.

A combination of biophysical and biochemical approaches was employed to probe the topology, arrangement, and function of the large surface subdomains of SGLT1 in living cells. Using atomic force microscopy on the single molecule level, Chinese hamster ovary cells overexpressing SGLT1 were probed with atomic force microscopy tips carrying antibodies against epitopes of different subdomains. Specific single molecule recognition events were observed with antibodies against loop 6-7, loop 8-9, and loop 13-14, demonstrating the extracellular orientation of these subdomains. The addition of D-glucose in Na+-containing medium decreased the binding probability of the loop 8-9 antibody, suggesting a transport-related conformational change in the region between amino acids 339 and 356. Transport studies with mutants C345A, C351A, C355A, or C361S supported a role for these amino acids in determining the affinity of SGLT1 for D-glucose. MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate and dithiothreitol inhibition patterns on alpha-methyl-glucoside uptake by COS-7 cells expressing C255A, C560A, or C608A suggested the presence of a disulfide bridge between Cys255 and Cys608. This assumption was corroborated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry showing mass differences in peptides derived from transporters biotinylated in the absence and presence of dithiothreitol. These results indicate that loop 6-7 and loop 13-14 are connected by a disulfide bridge. This bridge brings also loop 8-9 into close vicinity with the former subdomains to create a vestibule for sugar binding.

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies.

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.

Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells.

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substrate-carrier interactions of Na+-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na+ a sugar-binding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free L-glucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.

Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes.

Bacillus thuringiensis Cry delta-endotoxins cause death of susceptible insect larvae by forming lytic pores in the midgut epithelial cell membranes. The 65 kDa trypsin activated Cry4Ba toxin was previously shown to be capable of permeabilizing liposomes and forming ionic channels in receptor-free planar lipid bilayers. Here, magnetic ACmode (MACmode) atomic force microscopy (AFM) was used to characterize the lateral distribution and the native molecular structure of the Cry4Ba toxin in the membrane. Liposome fusion and the Langmuir-Blodgett technique were employed for supported lipid bilayer preparations. The toxin preferentially inserted in a self-assembled structure, rather than as a single monomeric molecule. In addition, the spontaneous insertion into receptor-free lipid bilayers lead to formation of characteristic pore-like structures with four-fold symmetry, suggesting that tetramers are the preferred oligomerization state of this toxin.

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment.

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease resistant-products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.