PubMed Health⌕ Search

Biomedical subjects

D Leckband

Publications and source records attributed to D Leckband.

11 recordsLinked to original sources
PubMed1998

Direct molecular level measurements of the electrostatic properties of a protein surface.

In this work, we used direct measurements with the surface force apparatus to determine the pH-dependent electrostatic charge density of a single binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumptions have not been tested directly at the molecular level. Using the force apparatus and immobilized, oriented monolayers of streptavidin, we measured a pI of 5-5.5 for the biotin-binding face of the protein. This differs from the pI of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson-Boltzmann equation, we then calculated the pH-dependent charge densities adjacent to the same face of the protein. These calculated values agreed quantitatively with those obtained by direct force measurements. Although our study focuses on the pH-dependence of surface electrostatics, this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbations that alter the charge distribution of the surfaces of immobilized molecules.

Binding Sites↗
PubMed1998

Molecular forces between membranes displaying neutral glycosphingolipids: evidence for carbohydrate attraction.

The surface force apparatus was used to determine the fundamental forces governing the adhesion between mixed bilayer membranes comprising lactosyl ceramide (LacCer) and di-tridecanoyl-phosphatidyl choline. Forces between membranes were quantified as a function of the glycolipid surface densities, which ranged from 0 to 30 mol %. Control measurements of the forces between pure phosphatidylcholine membranes and mixed bilayers of lactosyl ceramide with phosphocholine showed that the steric thickness of the carbohydrate headgroups increased from 19 to 25 A when the glycolipid density increased from 10 to 20 mol %. The layer compressibility also decreased with increasing carbohydrate coverage, but the corresponding adhesion between lactosyl ceramide-containing membranes increased with increasing amounts of glycosphingolipid in them. The nonspecific van der Waals forces accounted for the attraction measured in the control experiments and that between identical 10 mol % LacCer bilayers. However, the increase in the adhesion with increasing glycolipid density was 2-4 times greater than predicted by Lifschitz theory. Additionally, the forces measured during separation of membranes containing 20 and 30 mol % glycosphingolipid indicated that the headgroups bind and rearrange during bilayer detachment. The interactions between the carbohydrates are weak and apparently dynamic, and they generate an additional density-dependent intermembrane attraction that is on the order of the van der Waals force.

Adsorption↗
PubMed1997

Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains.

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

Bacterial Proteins↗
PubMed1995

Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis.

Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications. The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase. [3H]Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines. One of the two cysteines is surface accessible and lies in a hydrophilic environment while the other is in a hydrophobic environment. Chemical modification of the cysteines, both in the presence and in the absence of heparin, suggests that the surface-accessible cysteine lies in or near the active site of heparinase I. Preferential reactivity of this cysteine with negatively charged sulfhydryl-modifying reagents and the cysteines' high reactivity to iodoacetic acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region. The surface-accessible cysteine (cysteine-135) was mapped as the active-site cysteine by radiolabeling with [3H]iodoacetic acid and by tryptic digestion and peptide sequencing. Site-directed mutagenesis of cysteine-135 to a serine or an alanine in r-heparinase I demonstrates that this cysteine is essential for enzymatic activity. However, replacement of the surface-inaccessible cysteine by a serine or alanine has no effect.

Base Sequence↗
PubMed1995

The surface apparatus--a tool for probing molecular protein interactions.

The measurements described demonstrate the variety of protein or protein-mediated interactions that can be probed directly with the SFA. The distance, force and dynamic measurement capabilities provide tremendous potential for the identification and quantification of the molecular mechanisms and forces that both determine protein properties and influence protein function in diverse environments.

Electrochemistry↗
PubMed1995

Molecular mechanisms determining the strength of receptor-mediated intermembrane adhesion.

The strength of receptor-mediated cell adhesion is directly controlled by the mechanism of cohesive failure between the cell surface and underlying substrate. Unbinding can occur either at the locus of the specific bond or within the bilayer, which results in tearing the hydrophobic anchors from the membrane interior. In this work, the surface force apparatus has been used to investigate the relationship between the receptor-ligand bond affinities and the dominant mechanism of receptor-coupled membrane detachment. The receptors and ligands used in this study were membrane-bound streptavidin and biotin analogs, respectively, with solution affinities ranging over 10 orders of magnitude. With the optical technique of the surface force apparatus, the occurrence of membrane rupture was directly visualized in situ. The latter observations together with measurements of the corresponding intermembrane adhesive strengths were used to identify the dominant failure pathway for each streptavidin-analog pair. Even in cases where the membrane pull-out energy exceeded the equilibrium bond energy, cohesive failure occurred within the membrane interior at nearly all bond affinities considered. These results are consistent with previous findings and provide direct support for the commonly held view that, under nonequilibrium conditions of applied external stress, the gradient of the bond energy, not the equilibrium bond energy alone, determines the adhesive strength. Furthermore, our findings directly demonstrate that, in the presence of competing failure mechanisms, the preferred detachment mechanism- hence, the adhesive strength-will be determined by the bond that exhibits the weakest tensile strength. Because the tensile strength is determined by the gradient of the unbinding energy, the critical detachment force will be determined by both the bond energy and the effective bond length.

Animals↗
PubMed1994

Higher order self-assembly of vesicles by site-specific binding.

The association of lipid molecules into spherical vesicles in solution as a result of non-specific intermolecular forces constitutes a primary self-assembly process. Such vesicles can undergo a secondary self-assembly into higher order structures in a controlled and reversible manner by means of site-specific ligand-receptor (biotin-streptavidin) coupling. Cryoelectron microscopy shows these structures to be composed of tethered, rather than adhering, vesicles in their original, unstressed state. In contrast, vesicles aggregated by nonspecific, such as van der Waals, forces are deformed and stressed, producing unstable structures. Vesicle association by site-specific binding provides a practical mechanism for the production of stable, yet controllable, microstructured biomaterials.

Adhesiveness↗
PubMed1988

Function of tightly bound nucleotides on membrane-bound chloroplast coupling factor.

The kinetic behavior of tightly bound nucleotides on chloroplast coupling factor from spinach was determined under phosphorylating and nonphosphorylating conditions. Chloroplast coupling factor 1 (CF1) was labeled with tightly bound radioactive ADP and/or ATP at two specific sites and reconstituted with thylakoid membranes depleted of CF1 by treatment with NaBr. The initial incorporation and dissociation of ADP from one of the sites requires light but occurs at the same rate under phosphorylating and non-phosphorylating conditions. The initial rate is considerably slower than the rate of ATP synthesis, but nucleotide exchange is very rapid during steady-state ATP synthesis. A direct correspondence between this nucleotide binding site and a site on soluble CF1 that hydrolyzes ATP was demonstrated. A second site binds MgATP very tightly; the MgATP does not dissociate during ATP synthesis nor does its presence alter the rate of ATP synthesis. This is analogous to the behavior found for soluble CF1 during ATP hydrolysis. These results demonstrate that the tight-binding nucleotide sites on soluble CF1 and membrane-bound coupling factor are essentially identical in terms of binding properties and kinetic behavior during ATP hydrolysis and synthesis.

Adenosine Diphosphate↗
PubMed1987

Interactions between nucleotide binding sites on chloroplast coupling factor during ATP hydrolysis.

The initial hydrolysis of CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rat constant for the first-order process is independent of substrate concentration but increases hyperbolically to a limiting value of 0.43 s-1 with increasing concentrations of free Ca2+. A mechanism involving a Ca2+-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approximately 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s-1 as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changed. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

Adenosine Diphosphate↗