Biomedical subjects

H J Gruber

Publications and source records attributed to H J Gruber.

At least 19 recordsLinked to original sources

PubMed2005

Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.

Adsorption↗

PubMed2001

Spectrophotometric measurement of mercaptans with 4,4'-dithiodipyridine.

In a preceding study, 4,4'-dithiodipyridine (DTDP) was shown to be superior to 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) for spectrophotometric measurement of thiol groups in aqueous solution. (i) Sensitivity is higher because a larger absorbance increase is seen at a given thiol concentration. (ii) The intrinsic reactivity of thiolate anions for DTDP is much higher than for Ellman's reagent; thus, the reaction can be carried out at pH > or = 4.5 instead of at pH 8.0. In the present study, these advantages of DTDP were exploited for spectrophotometric measurement of thiols in organic solvent. DTDP was found to quantitatively react with nonpolar thiols when triethylamine was used as catalyst, intense light absorption (between 344 and 360 nm) was seen when the reaction was terminated with acetic acid, and the spectrophotometric responses were independent of the nonthiol portions of the mercaptans. The determination limit (10x the standard deviation of the reagent blank) was 3 microM, and the upper limit was approximately 40 microM on a typical spectrophotometer. The thiol contents of the mercaptans were independently verified by a modification of standard iodometry in which toluene/butanol or chloroform/butanol was included to dissolve nonpolar mercaptans.

Disulfides↗

PubMed2000

Immobilizing the moving parts of voltage-gated ion channels.

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

Base Sequence↗

PubMed1999

Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization.

A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure (strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial 'fluorescein-biotin', except that >10 h incubation times were required. The slow association of 'fluorescein-biotin' was attributed to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this study was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time.

Avidin↗

PubMed1999

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate.

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).

Avidin↗

PubMed1999

Detection of individual oligonucleotide pairing by single-molecule microscopy.

Hybridization of 20 mer probe oligonucleotides to complementary, surface-immobilized target oligonucleotides was visualized on a single-molecule basis by fluorescence microscopy. Coincident determination of the positions of both the target and the probe oligonucleotides using dual-wavelength fluorescence labeling allowed for highly reliable discrimination of specifically bound probe molecules from those being physisorbed. The figures of merit of the assay are characterized by the low probability for false positive (10(-4)) events and the high speed for detection of up to hundreds of different DNA fragments per second. The probability for false negative events is limited by the biochemical binding probability of short oligonucleotides. The potentials and limitations of this methodology for single-cell single-DNA analysis are discussed.

DNA Probes↗

PubMed1999

Single-molecule anisotropy imaging.

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

Anisotropy↗

PubMed1998

Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes.

Animals↗

PubMed1998

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Aluminum Silicates↗

PubMed1997

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor.

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.

Calcium Channels↗

PubMed1997

A non-invasive method for the tight anchoring of cells for scanning force microscopy.

Use of scanning force microscopy (SFM) for high resolution imaging of cell surfaces requires the cells to be tightly attached to substrates. Imaging of loosely adhered RBL-2H3 cells enabled determination of the cell size and investigation of larger structures and pseudopodia but failed in resolving more detail. Immobilization under non-invasive conditions via flexible crosslinkers containing a hydrophobic anchoring group enhanced resolution enormously. The cells were tightly attached to the substrates and were not removed by shear forces up to 80 nN as determined in a flow through apparatus. Morphological structures and dynamic processes on cell surfaces were observed as well as structural changes after cell stimulation upon ionomycin treatment. Molecular or atomic resolution, however, was not attainable which is attributed to the displacement of the flexible cell surface due to shear forces arising from the scanning tip during contact mode.

Animals↗

PubMed1997

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein.

The plasma membrane of rat erythrocytes contains a 47-kDa glycoprotein that binds the channel-forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N-terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N-termini of a group of membrane proteins that are thought to be involved in ADP-ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C-terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol-specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells' sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin-binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.

Animals↗

PubMed1996

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Animals↗

PubMed1996

Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.

Fluorescent Dyes↗

PubMed1995

Measurement of intravesicular volumes by salt entrapment.

Internal volume is a very sensitive parameter of vesicle morphology. Measurement of captured volumes by solute entrapment is legitimate for most types of vesicles (Perkin, W.R. et al. (1993) Chem. Phys. Lipids 64, 197-217). In this study chloride was selected as the most convenient marker ion because the ubiquity of Cl- in physiological buffers eliminates prelabeling with exogenous markers and because minute concentrations of trapped chloride are well detectable in the presence of large extravesicular nitrate concentrations. Perfect exchange of external chloride for nitrate was shown to be accomplished by gel filtration, dialysis, or sucrose gradient flotation-but only after significant technical improvements and/or elimination of experimental pitfalls. Reliability was cross-checked by simultaneous entrapment of Cl- and K+. Diafiltration and ion exchange chromatography appeared inapplicable for exchange of extravesicular salt. When a representative variety of vesicle preparations was analyzed for internal volume (as well as for external surface and size) unexpected features of vesicle morphology were discovered. This emphasizes the genuine role of macroscopic vesicle characterization in complementing information from electron microscopy.

Buffers↗

PubMed1994

External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods.

Three methods are presented for the determination of external surface of large lipid vesicles of different lamellarity with 2% absolute accuracy. These methods (referred to as EPR, NBD and TNBS assays) use different marker lipids which provide signals (electron paramagnetic resonance, fluorescence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) residues, and UV absorption increase of 2,4,6-trinitrobenzenesulfonic acid after reaction with aminolipids, respectively). The signals change upon addition of different membrane-impermeant reagents due to reaction with marker lipids at the external vesicle surface. They were applied to the same vesicle samples, including unilamellar and multilamellar vesicles, both at two different lipid compositions. External surface data matched for the three assays within 2%, but only after appropriate redesign or adaptation of so far published procedures. Main improvements related to slow influx of reagents (TNBS and NBD assays) or to redistribution of marker lipids (EPR assay), obscuring determination of outer vesicle surface from fast reaction between reagent and readily accessible marker lipids. Furthermore, suitable strategies were found to obtain accurate 100% values (reaction of all marker lipids present), required to relate external vesicle surface to total surface. This included corrections for light scattering (NBD assay) and for turbidity (TNBS assay). These three methods appear to close a gap in the methodology to determine external surface of vesicles for typical practical needs. In particular, the reliability range of the NBD assay could be extended to marker lipid densities as low as 1 marker lipid per 3000 lipids.

4-Chloro-7-nitrobenzofurazan↗

PubMed1994

Partial purification of the rat erythrocyte receptor for the channel-forming toxin aerolysin and reconstitution into planar lipid bilayers.

The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N-glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.

Animals↗

PubMed1988

Interaction of amphiphiles with integral membrane proteins. I. Structural destabilization of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols, and fatty amines.

The effect of model amphiphiles on the structural stability of the anion exchange protein (band 3) of the human erythrocyte membrane was studied by differential scanning calorimetry. The concentration of membranes, as well as the concentration, head group, alkyl chain length, degree of unsaturation, and double bond configuration of a variety of alkane derivatives were all varied in a systematic way. The depression of the denaturation temperature of band 3 per unit membrane concentration of the amphiphile was then determined in order to quantitate the potency of each drug. Saturated fatty acids of chain length C8 to C24 displayed a monotonic decrease in potency up to C20, followed by a dramatic diminution in potency at C22 and C24. Unsaturation caused only minor increases in the abilities of fatty acids to perturb the anion exchanger, and surprisingly, there was neither a trend for the number of double bonds nor a significant cis-trans distinction. Arachidonic acid, as an exception, was much more effective than any other amphiphile in destabilizing band 3. Fatty acids were about three times more potent than fatty amines and fatty alcohols; however, the enhanced partitioning of the latter into the membrane compensated at certain membrane/buffer ratios for its reduced intrinsic potency. A quantitative model interpretation of the data is presented in an accompanying paper.

Amines↗