Droplet sedimentation: A physical description and qualitative explanation of the phenomenon

Experiments with very simple geometric configurations and dynamic conditions were conducted to observe the formation of filaments and droplets from buoyant layers containing dense macromolecules. The development of the droplets into small vortex rings, and their motions were followed. The observations are described. These experiments were conducted in an effort to understand and to predict streaming, or tumbling, or droplet sedimentation in centrifuges. A sequence of physical processes composing the phenomenon of droplet sedimentation is proposed and discussed.     

Effect of ionic strength on the diffusion coefficient of chondroitin sulfate and heparin measured by quasielastic light scattering

The normal modes for a mixture of charged macromolecules and electrolyte solution are calculated. We derive a generalized Debye relaxation time and the apparent diffusion coefficient of the macroion, which is shown to increase from its Stokes value, obtained in excess of added salt, with decreasing ionic strength. We test our result with experimental data for macromolecules with different charge densities: heparin and chondroitin sulfate. Besides, we show for this latter molecule that while the diffusion coefficient is increased, the scattered intensity is decreased but not by the same factor. Our results are compared with other theories developed in quasielastic light scattering.     

Electrostatic interactions across a charged lipid bilayer

We present theoretical work in which the degree of electrostatic coupling across a charged lipid bilayer in aqueous solution is analyzed on the basis of nonlinear Poisson–Boltzmann theory. In particular, we consider the electrostatic interaction of a single, large macroion with the two apposed leaflets of an oppositely charged lipid bilayer where the macroion is allowed to optimize its distance to the membrane. Three regimes are identified: a weak and a high macroion charge regime, separated by a regime of close macroion–membrane contact for intermediate charge densities. The corresponding free energies are used to estimate the degree of electrostatic coupling in a lamellar cationic lipid–DNA complex. That is, we calculate to what extent the one-dimensional DNA arrays in a sandwich-like lipoplex interact across the cationic membranes. We find that, in spite of the low dielectric constant inside a lipid membranes, there can be a significant electrostatic contribution to the experimentally observed cross-bilayer orientational ordering of the DNA arrays. Our approximate analytical model is complemented and supported by numerical calculations of the electrostatic potentials and free energies of the lamellar lipoplex geometry. To this end, we solve the nonlinear Poisson–Boltzmann equation within a unit cell of the lamellar lipoplex using a new lattice Boltzmann method. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Positively charged surfaces increase the flexibility of DNA

Many proteins "bind" DNA through positively charged amino acids on their surfaces. However, to overcome significant energetic and topological obstacles, proteins that bend or package DNA might also modulate the stiffness that is generated by repulsions between phosphates within DNA. Much previous work describes how ions change the flexibility of DNA in solution, but when considering macromolecules such as chromatin in which the DNA contacts the nucleosome core each turn of the double helix, it may be more appropriate to assess the flexibility of DNA on charged surfaces. Mica coated with positively charged molecules is a convenient substrate upon which the flexibility of DNA may be directly measured with a scanning force microscope. In the experiments described below, the flexibility of DNA increased as much as fivefold depending on the concentration and type of polyamine used to coat mica. Using theory that relates charge neutralization to flexibility, we predict that phosphate repulsions were attenuated by approximately 50% in the most flexible DNA observed. This simple method is an important tool for investigating the physiochemical causes and molecular biological effects of DNA flexibility, which affects DNA biochemistry ranging from chromatin stability to viral encapsulation.     

Domain formation induced by the adsorption of charged proteins on mixed lipid membranes

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation.     

Integral equation theories for predicting water structure around molecules

Water plays a crucial role in the structure and function of proteins and other biological macromolecules; thus, theories of aqueous solvation for these molecules are of great importance. However, water is a complex solvent whose properties are still not completely understood. Statistical mechanical integral equation theories predict the density distribution of water molecules around a solute so that all particles are fully represented and thus potentially both molecular and macroscopic properties are included. Here we discuss how several theoretical tools we have developed have been integrated into an integral equation theory designed for globular macromolecular solutes such as proteins. Our approach predicts the three-dimensional spatial and orientational distribution of water molecules around a solute. Beginning with a three-dimensional Ornstein-Zernike equation, a separation is made between a reference part dependent only on the spatial distribution of solvent and a perturbation part dependent also on the orientational distribution of solvent. The spatial part is treated at a molecular level by a modified hypernetted chain closure whereas the orientational part is treated as a Boltzmann prefactor using a quasi-continuum theory we developed for solvation of simple ions. A potential energy function for water molecules is also needed and the sticky dipole models of water, such as our recently developed soft-sticky dipole (SSD) model, are ideal for the proposed separation. Moreover, SSD water is as good as or better than three point models typically used for simulations of biological macromolecules in structural, dielectric and dynamics properties and yet is seven times faster in Monte Carlo and four times faster in molecular dynamics simulations. Since our integral equation theory accurately predicts results from Monte Carlo simulations for solvation of a variety of test cases from a single water or ion to ice-like clusters and ion pairs, the application of this theory to biological macromolecules is promising.     

Surface charging by large multivalent molecules. Extending the standard Gouy-Chapman treatment.

Traditionally, Gouy-Chapman theory has been used to calculate the distribution of ions in the diffuse layer next to a charged surface. In recent years, the same theory has found application to adsorption (incorporation, partitioning) of charged peptides, hormones, or drugs at the membrane-water interface. Empirically it has been found that an effective charge, smaller than the physical charge, must often be used in the Gouy-Chapman formula. In addition, the large size of these molecules can be expected to influence their adsorption isotherms. To improve evaluation techniques for such experiments, comparatively simple extensions of the standard Gouy-Chapman formalism have been studied which are based on a discrete charge virial expansion. The model allows for the mobility of charged groups at the interface. It accounts for finite size of the adsorbed macromolecules and for discrete charge effects arising from pair interactions in the interface plane. In contrast to previous discrete charge treatments this model nearly coincides with the Gouy-Chapman formalism in the case where the adsorbing molecules are univalent. Large discrepancies are found for multivalent molecules. This could explain the reduced effective charges needed in the standard Gouy-Chapman treatment. The reduction factor can be predicted. The model is mainly limited to low surface coverage, typical for the adsorption studies in question.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

Eliminating the interferences from TRIS buffer and SDS in protein analysis by fused-droplet electrospray ionization mass spectrometry

Multiply charged protein ions were detected from the solutions containing a high concentration of tris(hydroxymethyl) aminomethane buffer (TRIS) and sodium dodecyl sulfate (SDS) using fused-droplet electrospray ionization mass spectrometry (FD-ESI/MS). The sample aerosols were generated at ambient temperature with a pneumatic nebulizer commonly used to produce sample aerosols in an atmospheric pressure chemical ionization (APCI) source. The aerosols were carried by nitrogen gas to the tip of a capillary where charged methanol droplets had been continuously generated by electrospraying an acidic methanol solution. The neutral sample aerosols then fused with the charged methanol droplets and electrospray ionization proceeded from the newly formed fused droplets to generate multiply charged protein ions. Because of its low solubility in methanol, TRIS molecules (concentration as high as 1 M) were efficiently excluded from the newly formed droplets and the protein ion signals were detected and observed in the mass spectra. To remove the interferences from SDS, equal moles of positively charged cetyltrimethylammonium bromide (CTAB) was added into the SDS containing sample solution to form the dodecyl sulfate-cetyltrimethylammonium ion pair (DS-CTA). The DS-CTA ion pair has a low polarity and solubility in methanol and is excluded from the fused droplet. Protein ions were still detected from the solution containing 10(-2) M of SDS.     

Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry

The extension of mass spectrometry to high molecular weight biopolymers based upon electrospray ionization and the on-line combination with capillary electrophoresis is described. Electrospray ionization produces gas-phase intact multiply charged molecular ions of biomolecules from highly charged liquid droplets by a high electric field. For high molecular weight substances electrospray ionization results in a characteristic bell-shaped distribution of multiply charged ions, with each adjacent major peak in the spectrum differing by one charge. Multiply charged molecular ions of proteins with molecular weights greater than 130,000 have been observed with a quadrupole mass spectrometer of limited mass-to-charge range (m/z 1700). Molecular weights can be readily determined for large proteins with accuracies in the range of +/- 0.01 to 0.05%; at least an order of magnitude further improvement appears feasible with improved techniques and instrumentation. The electrospray ionization method is sensitive, presently requiring samples in the 100 fmol to 10 pmol range for proteins. Initial results combining rapid separations by capillary zone electrophoresis with on-line mass spectrometric detection via the electrospray ionization source are demonstrated for myoglobin and other proteins and polypeptides. The potential for extension of these methods to molecular weights on the order of 10(6) is discussed.     

Impact of Cl- and Na+ ions on simulated structure and dynamics of betaARK1 PH domain

A nonzero net charge of proteins at pH 7 is usually compensated by the addition of charge-balancing counter ions during molecular dynamics simulation, which reduces electrostatic interactions. For highly charged proteins, like the betaARK1 PH domain used here, it seems reasonable to also add explicit salt ions. To assess the impact of explicit salt ions, two molecular dynamics simulations of solvated betaARK1 PH domain have been carried out with different numbers of Cl- and Na+ ions, based on the Cornell et al. force field and the Ewald summation, which was used in the treatment of long-range electrostatic interactions. Initial positions of ions were obtained from the AMBER CION program. Increasing the number of ions alters the average structure in loop regions, as well as the fluctuation amplitudes of dihedral angles. We found unnaturally strong interactions between side chains in the absence of salt ions. The presence of salt ions reduces these electrostatic interactions. The time needed for the equilibration of the ionic environment around the protein, after initial placement of ions close to oppositely charged side chains, is in the nanosecond time range, which can be shortened by using a higher ionic strength. Our results also suggest selecting those methods that do not place the ions initially close to the protein surface.     

MALDI-QTOFMS/MS identification of glycoforms from the urine of a CDG patient

Identification of single glycoconjugate components in a complex mixture from the urine of a patient suffering from a congenital disorder of glycosylation was probed by MALDIMS analysis on a hybrid quadrupole time-of-flight instrument. In negative ion mode, complex maps containing more than 50 ionic species were obtained and a number of molecular ions directly as-signed using a previously developed computer-assisted algorithm. To confirm the data and determine the carbohydrate sequence, single molecular ions were selected and submitted to fragmentation experiments. Interpretation of fragmentation spectra was also assisted by the soft-ware using alignment with spectra generated in silico. According to fragmentation data, the majority of glycoconjugate ionic species could be assigned to free oligosaccharides along with ten species tentatively assigned to glycopeptides. Following this approach for glycan identification by a combination of MALDI-QTOFMS and MS/MS experiments, computer-assisted assignment and fragment analysis, data for a potential glycan data base are produced. Of high benefit for this approach are two main factors: low sample consumption due to the high sensitivity of ion formation, and generation of only singly charged species in MALDIMS allowing interpretation with-out any deconvolution. In this experimental set-up, sequencing of single components from the MALDI maps by low energy CID followed by computer-assisted assignment and data base search is proposed as a most efficient strategy for the rapid identification of complex carbohydrate structures in clinical glycomics.     

An ion switch regulates fusion of charged membranes

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

A kinetic role for ionizable sites in membrane channel proteins

Electrically charged residues in a membrane channel protein will certainly have a direct effect upon its gating and selectivity if they are near the channel pore. It is customary to regard the charged state of such residues as a fixed feature of the channel. In this paper it is argued that far from being fixed, the charged state of ionizable residues near the pore will very probably change rapidly in response to the channel opening and to ions passing through it. Calculations are presented using simple models which demonstrate that changes in the dielectric environment and changes in the distances to other charged groups resulting from channel opening can shift the effective pK values of the sites by 3 or 4 units leading to switching of its charged state. Examples are given of how this time dependent charge state of ionizable residues may play an important role in the functioning of channels. Also, by considering the influence of the electric field due to the mobile ion upon the charge state of a residue in the channel wall, it is shown that a channel lined with acid residues may very effectively block the passage of cations while allowing the passage of anions.     

Effects of Hydration on Steric and Electric Charge-Induced Interstitial Volume Exclusion—a Model

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

A photographic technique for investigating the splashing of water drops on leaves

The formation of splash from water drops falling onto bean leaves was investigated by multiple exposure flash photography. A relatively simple technique was developed which recorded up to four images at 2 ms intervals, of a splash on one photograph. The images were recorded on colour reversal film, and the flash sequence was colour coded to aid the interpretion of the superimposed images. Analysis of the photographs allowed measurement of the initial speed and angle of ejection of different sized splash droplets. Droplets were ejected at speeds of up to about 10 m s^-1although most speeds were less than about 3 m s^-1, and small droplets tended to travel faster than large ones. Droplets were ejected at all angles but the majority were ejected almost horizontally; there was little correlation between droplet size and angle of ejection. Deposition patterns estimated from calculated droplet trajectories compared well with measured ones.