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Biomedical subjects

W Knoll

Publications and source records attributed to W Knoll.

At least 55 records · Page 3Linked to original sources
PubMed1994

Direct force measurements of specific and nonspecific protein interactions.

Streptavidin-biotin (receptor-ligand) interaction forces were measured directly as a function of their intermolecular separation in various salt solutions and at various temperatures with a surface forces apparatus. Electrostatic and van der Waals forces were found to dominate the long-range streptavidin-biotin interaction at > 20 A. At intermediate separations, down to approximately 10 A, the interaction is governed by repulsive steric and attractive van der Waals and hydrophobic forces. A much stronger short-range attraction giving rise to the strong, specific adhesive binding was measured at molecular separations of less than 5 A. A decrease in the pH from 7.2 to 6.0 resulted in complete charge reversal on the binding surface of streptavidin (pK approximately 6) from net negative to net positive, while leaving the negatively charged biotin surface (pK approximately 3.0) unchanged, and the long-range interaction switched from repulsive to attractive. This observed behavior can be attributed to the titration of two histidines on the biotin binding surface of streptavidin. These results reveal a strong sensitivity of the long-range interaction forces to the detailed amino acid composition of the biotin binding surface. They also demonstrate the powerful regulatory potential conferred by small changes in local surface ionic conditions on protein interaction forces over different distance regimes. The effects of temperature on receptor-ligand dynamics and on the strength of intermembrane adhesion forces were studied by measuring the long-range force profiles and short-range adhesion forces above and below the chain melting temperature (Tc approximately 30 degrees C) of the lipids in the supporting bilayers. Increased bilayer fluidity due to a temperature increase to 33 degrees C (T > Tc) increased short-range adhesion by 7-fold relative to bilayers in the gel state at 25 degrees C (T < Tc). This effect was attributed to the enhanced rates of lateral diffusion and molecular rearrangements on the more fluid bilayer surfaces, which resulted in greater and more rapid intermembrane bond formation. A change in the rates of molecular rearrangements was also found to affect the repulsive part of the interaction potential at intermediate separations (10-20 A) via modulation of the steric repulsion between streptavidin and the highly flexible, polymer-like biotin molecules. This is expected to have a large effect on the association rates of receptor-ligand binding, even if it does not change the equilibrium binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1994

Formation of protein multilayers and their competitive replacement based on self-assembled biotinylated phospholipids.

Based on specific recognition processes the build-up of protein multilayers was achieved using streptavidin layers as a docking matrix. For this purpose, streptavidin was organized at biotin-containing monolayers, liposomes, and self-assembled layers on gold. Thus, mixed double and triple layers of streptavidin, Con A, Fab fragments, and hormones were prepared and characterized by fluorescence microscopy and plasmon spectroscopy. Using biotin analogues with lower binding constants several cycles of multilayer formation followed by competitive replacement could be achieved.

Bacterial Proteins↗
PubMed1993

Attempts to mimic docking processes of the immune system: recognition-induced formation of protein multilayers.

The assemblage of protein multilayers induced by molecular recognition, as seen, for example, in the immune cascade, has been mimicked by using streptavidin as a docking matrix. For these experiments, this protein matrix was organized on liposomes, monolayers at the air-water interface, and self-assembled layers on gold, all three containing biotin lipids. The docking of streptavidin to biotin at liposomal surfaces was confirmed by circular dichroism. Mixed double and triple layers of streptavidin, concanavalin A, antibody Fab fragments, and hormones are prepared at the air-water interface and on gold surfaces and were characterized by fluorescence microscopy and plasmon spectroscopy. With the use of biotin analogs that have lower binding constants it has been possible to achieve multiple formation and competitive replacement of the oriented protein assemblages.

Bacterial Proteins↗
PubMed1993

Ca(2+)-induced lateral phase separation in black lipid membranes and its coupling to the ion translocation by gramicidin.

We analyze the single-channel current fluctuations of gramicidin incorporated into biomolecular lipid membranes (BLM) of binary mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) as a function of the Ca2+ concentration in the electrolyte (0.5 M CsCl, pH 6) solution. At low Ca2+ levels (cCa2+ < 10(-6) M) a monomodal conductance histogram and a single average lifetime suggests a homogeneous mixture over the full range of composition (PG(1-x)PCx, 0 < or = x < or = 1). At higher Ca2+ concentrations phase separation processes are inferred from the appearance of bimodal conductance histograms. The two channel populations (in the two coexisting phases) can also be distinguished through their different average lifetimes. By a systematic variation of the mole fractions of the two lipid components we derive the respective phase boundaries and thus the full Ca2+ concentration-composition phase diagram.

Calcium↗
PubMed1992

Long-range attraction and molecular rearrangements in receptor-ligand interactions.

A surface force apparatus was used to measure a long-range attractive protein-ligand force at separations D less than 85 angstroms. This force may effectively "steer" ligand trajectories, resulting in a greater than 27-fold enhancement of the association rate. A much stronger specific attraction is measured at contact (D less than 4 angstroms). A sevenfold increase in intermembrane adhesion resulted from increased lateral mobility of the receptors and molecular rearrangements in membranes above the solid-fluid transition temperature.

Bacterial Proteins↗
PubMed1992

Streptavidin binding observed with an atomic force microscope.

An atomic force microscope (AFM) was used to investigate a specific recognition reaction: the binding of streptavidin to a biotinylated lipid bilayer. Prior to the recognition reaction, the phase coexistence of the lipid bilayer was clearly observed: fluid domains were lower than the crystalline domains. After introducing to the bilayer a very dilute solution of streptavidin to give a final concentration of approximately 0.5 microM, the recognition reaction was imaged in real time. Several hours later, we observed a contrast reversal, i.e., the previously lower fluid domains grew so much in height that they became higher than the crystalline domains. We found that the streptavidin molecules bound almost exclusively to the biotin in the fluid domain (less than 0.25% coverage of the crystalline domains). The apparent structure of the few streptavidin molecules bound to the crystalline domain of the bilayer is shown to depend on the applied force. Finally, in a 2-dimensional quasi-crystal in which the streptavidin molecules were compressed at the air-water interface molecular resolution was achieved.

Bacterial Proteins↗
PubMed1992

Streptavidin binding to biotinylated lipid layers on solid supports. A neutron reflection and surface plasmon optical study.

Neutron reflection and surface plasmon optical experiments have been performed to evaluate structural data of the interfacial binding reaction between the protein streptavidin and a solid-supported lipid monolayer partly functionalized by biotin moieties. Since both experimental techniques operate in a total internal reflection geometry at a substrate/solution interface, identical sample architectures allow for a direct comparison between the results obtained with these two recently developed methods. It is found that a monomolecular layer of dipalmitoyllecithin doped with 5 mol% of a biotinylated-phosphatidylethanolamine shows a thickness of d1 approximately (3.4 +/- 0.5) nm. Binding of streptavidin to the biotin groups results in an overall layer thickness of d = (5.9 + 0.5) nm that demonstrates the formation of a well-ordered protein monolayer with the (biotin+spacer) units of the functionalized lipids being fully embedded into the binding pocket of the proteins. It is demonstrated by model calculations that a more detailed picture of the internal structure of this supramolecular assembly can only be obtained if one uses deuterated lipid molecules, thus generating a high contrast between individual layers.

Bacterial Proteins↗
PubMed1991

Measurement of ligand-receptor interactions.

One distinguishing feature of "life" is that the physical forces between biological molecules and membrane surfaces are often highly specific, in contrast to nonspecific interactions such as van der Waals, hydrophobic, and electrostatic (Coulombic) forces. We have used the surface-forces-apparatus technique to study the specific "lock and key" or "ligand-receptor" interaction between two model biomembrane surfaces in aqueous solution. The membranes were lipid bilayers supported on mica surfaces; one carrying streptavidin receptors, the other exposing biotin ligand groups. We found that, although no unusual or specific interaction occurs between two avidin or two biotin surfaces, an avidin and a biotin surface exhibit a very strong, very short-range (less than 1 nm) attraction and that the binding mechanism involves equally specific molecular rearrangements. The results also show that highly specific biological interactions such as are involved in immunological recognition and cell-cell contacts may be studied at the molecular level and in real time by the surface-forces-apparatus technique.

Aluminum Silicates↗
PubMed1991

Lateral order in binary lipid alloys and its coupling to membrane functions.

Densitometry, Raman spectroscopy and small angle neutron scattering are employed to elucidate the miscibility behavior of lipid mixtures organized as liposomal dispersions. First, temperature-composition-phase diagrams for several binary alloys of dialkyl-lecithins differing in chain lengths by an increasing number of CH2-groups are derived. A mixture of dimyristoyllecithin and distearoyllecithin (delta CH2 = 4) shows a peritectic phase behavior with a miscibility gap in the gel state. In the fluid phase, at high enough temperatures, homogeneous mixtures of the two components are formed at all molar ratios. However, upon approaching a critical point by either lowering the temperature or increasing the hydrostatic pressure, critical concentration fluctuations are observed. If one component of a binary mixture is charged, electrostatic interactions can be used to induce phase separation at constant temperature. This is demonstrated for Ca2(+)-driven demixing in alloys of lecithin and negatively charged phosphatidylglycerol. The influence of the various concepts for the induction of lateral structure formation in lipid membranes on integral functional units like ionophores is demonstrated by analysing the single channel current fluctuations of gramicidin in bimolecular lipid membranes. Ca2+, as well as polyelectrolyte-induced phase separations are shown and discussed as examples for the important (lateral) order-function relationship in biomembranes.

Electrochemistry↗
PubMed1990

pH-control of the miscibility properties of a binary lipid alloy and its influence on the ion transport by gramicidin.

We studied the coupling of a membrane function (the transport of ions by the pore forming polypeptide gramicidin) to chemically driven phase changes in black membranes of binary lipid mixtures. In particular, we investigated the influence of the aqueous pH value on the fluid-fluid demixing effect of Ca2+ to phosphatidylcholine/phosphatidylglycerol bilayers. It is found that one can switch, under certain conditions, between a homogeneously mixed and a phase separated membrane by changing the pH. We interpret this as being caused by the change in the degree of dissociation of one of the lipid components.

Electric Conductivity↗
PubMed1989

Phase separation in bimolecular mixed lipid membranes induced by polylysine.

We demonstrate, for the first time, polylysine-induced phase separation in a bimolecular lipid membrane of a lecithin/phosphatidylglycerol-mixture by analysing the single channel current fluctuations of gramicidin. The bimodal conductance histograms are direct evidence for the incorporation of the transport system into the two coexisting phases of different composition.

Dimyristoylphosphatidylcholine↗
PubMed1989

Lateral order in mixed lipid bilayers and its influence on ion translocation by gramicidin: a model for the structure-function relationship in membranes.

The temperature-composition phase diagram of dimyristoylphosphatidylcholine and dipentadecylphosphatidylglycerol (DiC15PG) was determined by mass densitometry. For a mixture containing 30 mol% DiC15PG, the homogeneous distribution of the 2 components is demonstrated in the fluid state at T = 35 degrees C by small-angle neutron scattering in combination with the inverse contrast variation method. By the same technique, the coexistence of fluid and condensed phases at T = 23.3 degrees C could be shown in agreement with the densitometric data. Furthermore, it is demonstrated that Ca++ induces, even at T = 35 degrees C, separation into 2 fluid phases. A corresponding phase separation is found in bimolecular lipid membranes ("black films") by analysis of the single-channel conductance fluctuations of gramicidin A incorporated into an equimolarly mixed membrane of neutral lecithin and charged phosphatidic acid. The results are discussed as primary examples on the model-membrane level for the important structure-function relationship of biomembranes.

Calcium↗