Ion hydration: Implications for cellular function, polyelectrolytes, and protein crystallization

Only oppositely charged ions with matching absolute free energies of hydration spontaneously form inner sphere ion pairs in free solution [K.D.Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311.]. We approximate this with a Law of Matching Water Affinities which is used to examine the issues of (1) how ions are selected to be compatible with the high solubility requirements of cytosolic components; (2) how cytosolic components tend to interact weakly, so that association or dissociation can be driven by environmental signals; (3) how polyelectrolytes (nucleic acids) differ from isolated charges (in proteins); (4) how ions, osmolytes and polymers are used to crystallize proteins; and (5) how the "chelate effect" is used by macromolecules to bind ions at specific sites even when there is a mismatch in water affinity between the ion and the macromolecular ligands.     

Size-dependent mobile surface charge model of cell electrophoresis

A model that accurately predicts the effects of cellular size and electric field strength on electrophoretic mobility has been developed. Previous models have predicted that electrophoretic mobility (EPM) is dependent only on cell surface charge, bath viscosity and ionic strength of the electrolyte. However, careful analysis of experimental data from the literature shows that these models do not accurately depict the relationship between chemically determined surface charge and observed mobility. We propose a new model that accounts for electrically driven redistribution of mobile surface charge islands, such as the recently proposed lipid raft structures. This model predicts electrophoretic mobility as a function of a new dimensionless quantity, A, that incorporates the cell radius, the electric field strength, and the average diameter of charged membrane complexes.     

Endocytosis of liposomes bound to cell surface proteins measured by flow cytofluorometry.

A new technique for the quantification of cellular receptor-mediated endocytosis has been developed based on the analysis by flow cytometry of ligand-bearing liposomes containing the fluorochrome carboxyfluorescein. Carboxyfluorescein encapsulated at high concentrations in protein A-bearing liposomes is self-quenched. Binding and internalization of such liposomes by cells via antibodies directed towards membrane surface determinants results in the release of the liposome-encapsulated carboxyfluorescein into the cytoplasm causing an increase in cell-associated fluorescence. This increase can be quantified on a flow cytofluorometer.     

Conformation and packing analysis of polysaccharides and derivatives. I: Mannan

A previously described procedure for simultaneous optimization of bond lengths and angles was used to test different models for mannan I. Potential hydrogen bonds and the glycosidic angle were included in the optimization. A conformational model with bifurcated intramolecular hydrogen bonds of the type observed in the methyl cellobioside methanol complex showed the best agreement with available exprerimental data. The coordinates of this model were provided by computer calculations. The available X-ray data, however, were not sufficient for selecting this model; rather, ir data were necessary to furnish the needed information. The different conformational models tested all showed an almost constant virtual bond length O(1)–O(4) of the β-pyranose residue. This was in contrast to the previously obtained results for the α-pyranose residues.     

Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors.

Steric effects can influence the binding of a cell surface receptor to a multivalent ligand. To account for steric effects arising from the size of a receptor and from the spacing of binding sites on a ligand, we extend a standard mathematical model for ligand-receptor interactions by introducing a steric hindrance factor. This factor gives the fraction of unbound ligand sites that are accessible to receptors, and thus available for binding, as a function of ligand site occupancy. We derive expressions for the steric hindrance factor for various cases in which the receptor covers a compact region on the ligand surface and the ligand expresses sites that are distributed regularly or randomly in one or two dimensions. These expressions are relevant for ligands such as linear polymers, proteins, and viruses. We also present numerical algorithms that can be used to calculate steric hindrance factors for other cases. These theoretical results allow us to quantify the effects of steric hindrance on ligand-receptor kinetics and equilibria.     

Conformation of coenzyme fragments when bound to lactate dehydrogenase

The conformations of adenosine, 5′-AMP and 5′-ADP when bound to dogfish M₄ lactate dehydrogenase at pH 7.8 or greater have been determined at 2.8 Å resolution to investigate the events on coenzyme binding. The coenzyme fragments AMP and ADP induce a conformational change in lactate dehydrogenase at pH values less than 6.0 in the same way as do NAD⁺, NADH or ADPR at any pH value. The structure of NAD⁺ when bound to lactate dehydrogenase had previously been determined at 5.0 Å resolution. The structures of the bound adenosine, AMP, ADP and NAD⁺ are compared with the preliminary structure of NAD in a 3.0 Å resolution map of the ternary complex LDH-NAD—pyruvate. Small but significant changes in the binding of the phosphates could be important in the folding of the protein loop over the substrate binding pocket.     

Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibody.

We present a joint theoretical and experimental study on the effects of competition for ligand between receptors in solution and receptors on cell surfaces. We focus on the following experiment. After ligand and cell surface receptors equilibrate, solution receptors are introduced, and the dissociation of surface bound ligand is monitored. We derive theoretical expressions for the dissociation rate and compare with experiment. In a standard dissociation experiment (no solution receptors present) dissociation may be slowed by rebinding, i.e., at high receptor densities a ligand that dissociates from one receptor may rebind to other receptors before separating from the cell. Our theory predicts that rebinding will be prevented when S much greater than N2Kon/(16 pi 2D a4), where S is the free receptor site concentration in solution, N the number of free surface receptor sites per cell, Kon the forward rate constant for ligand-receptor binding in solution, D the diffusion coefficient of the ligand, and a the cell radius. The predicted concentration of solution receptors needed to prevent rebinding is proportional to the square of the cell surface receptor density. The experimental system used in these studies consists of a monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), that reversibly binds to a monoclonal anti-DNP immunoglobulin E (IgE). This IgE is both a solution receptor and, when anchored to its high affinity Fc epsilon receptor on rat basophilic leukemia (RBL) cells, a surface receptor. For RBL cells with 6 x 10(5) binding sites per cell, our theory predicts that to prevent DCT rebinding to cell surface IgE during dissociation requires S much greater than 2,400 nM. We show that for S = 200-1,700 nM, the dissociation rate of DCT from surface IgE is substantially slower than from solution IgE where no rebinding occurs. Other predictions are also tested and shown to be consistent with experiment.     

Conformation of histidine model peptides. I. Conformational energy calculations for L-alanyl-L-histidine diketopiperazine

Semiempirical conformational energy calculations were carried out for the cyclic dipeptide L -alanyl-L -histidine diketopiperazine. The results indicate that electrostatic effects are probably significant in determining the conformation assumed by this molecule. When the imidazole group is in its uncharged state the most stable conformations of the molecule are those with the imidazole ring folded over the diketopiperazine ring (χ¹ = 60°). Upon protonation of the imidazole group the folded conformation may be destabilized relative to conformations characterized by χ¹ positions near 180°.     

Dielectric properties of polyelectrolytes. I. A study on tetra-n-butyl ammonium polyacrylate

Dielectric constant and loss of aqueous solutions of tetra-n-butyl ammonium polyacrylate ((Bu)₄NPA) were measured in the frequency range from 300 Hz to 6 MHz as compared with sodium and other salts at various conditions. Our results show that there are two dispersions observed in the low-frequency range (LFD, several ten kHz to MHz), respectively, both of which are roughly expressed as the Cole-Cole dispersion formula with Cole parameters about 0.3. The large values of dielectric increment, its nonlinear concentration dependence, and other features suggest that both dispersions are explained by relaxations of two different ionic processes. For HFD, experimental results were qualitatively similar to those have been reported and compared with theories of the Maxwell-Wagner-type effect. On the other hand, LFD may be attributable to the polarization of loosely bound counterions. A model available for LFD was presented on the basis of counterion fluctuation.     

Orientation of bound ligands in mannose-binding proteins. Implications for multivalent ligand recognition

Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.     

Conformation of a bound inhibitor of blood coagulant factor Xa

13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.     

Conformation of bovine myelin basic protein purified with bound lipids

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having α-helix and β-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results. Received: 13 November 1998 / Accepted: 1 February 1999