A natural and readily available crowding agent: NMR studies of proteins in hen egg white

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media.     

Direct discrimination between models of protein activation by single-molecule force measurements

The limitations imposed on the analyses of complex chemical and biological systems by ensemble averaging can be overcome by single-molecule experiments. Here, we used a single-molecule technique to discriminate between two generally accepted mechanisms of a key biological process--the activation of proteins by molecular effectors. The two mechanisms, namely induced-fit and population-shift, are normally difficult to discriminate by ensemble approaches. As a model, we focused on the interaction between the nuclear transport effector, RanBP1, and two related complexes consisting of the nuclear import receptor, importin beta, and the GDP- or GppNHp-bound forms of the small GTPase, Ran. We found that recognition by the effector proceeds through either an induced-fit or a population-shift mechanism, depending on the substrate, and that the two mechanisms can be differentiated by the data.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

Use of immobilized lactoperoxidase to label L cell proteins involved in adhesion to polystyrene

Proteins involved in the attachment of murine L cells to polystyrene have been identified by a technique designed to iodinate only those macromolecules coming into closet apposition to the substratum. Whereas soluble lactoperoxidase (LPO) catalyzes the radioiodination of a broad spectrum of polypeptides, the same enzyme immobilized on polystyrene tissue culture flasks discriminately labels 55,000 and 42,000 mol wt polypeptides that adhere tightly to the substratum after the cells are removed. One-dimensional peptide mapping following limited proteolysis showed that the labeled 55,000 mol wt polypeptide is similar to a component of comparable molecular weight present in the detergent- extracted cytoskeleton. The functional association of two cytoskeletal structures, presumably 10-nm filaments and actin, is discussed, and alternative explanations for their susceptibility to iodination by immobilized LPO are presented.     

Mechanisms common to biological macromolecules and gels

Functional Biological macromolecules arising from folding, cross-connection and solvation of long chain biopolymers forming three-dimensional networks may be regarded as Gels. Both involve identical internal competitive forces that are selectively influenced by external conditions and conspire to adjust conformations and modulate activities. In spite of important differences in size, chemical composition, polymer bonding, density and configuration, biological macromolecules indeed manifest some of the essential physical-chemical properties of gels when involved in equilibria and rate processes. This result represents a presumptive evidence for common underlying mechanisms in functional molecules and gels. Thus, the present and highly perfectible model explains why and how, depending on initial conditions, a system may respond differently to an external parameter, and similarly to different parameters. Moreover, the fact that any localized change in one of the competitive forces gives to a pressure in the system as a whole provides an explanation for the mechanism of the transmission of information.     

Origin of low-frequency motions in biological macromolecules. A view of recent progress in the quasi-continuity model

The recent progress in the quasi-continuity model and its applications in studying the low-frequency internal motions of biological macromolecules have been surveyed. Emphasis is placed on revealing the origin of this kind of internal collective motion, which involves many atoms and has significant biological functions. In light of such a line, the low-frequency motions in alpha-helix structure, beta-structure (including beta-sheet and beta-barrel), and DNA double-helix structure, the three most fundamental component elements in biological macromolecules, are discussed, and the corresponding physical pictures described. It turns out that the low-frequency motion in biological macromolecules originates from their two common intrinsic characteristics, i.e., they possess a series of weak bonds, such as hydrogen bonds and salt bridges, and a substantial mass distributed over the region containing those weak bonds.     

Mucus glycoproteins from ''normal'' human tracheobronchial secretion.

Mucous secretions were collected from tracheas of patients undergoing minor surgery under general anaesthesia with tracheal intubation, and mucus glycoproteins were isolated by using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. 'Whole' mucins were excluded from a Sepharose CL-2B gel, whereas subunits obtained after reduction were included. Trypsin digestion of subunits afforded high-Mr glycopeptides (T-domains), which were further included in the gel. The latter fragments are heterogeneous and comprise two or three populations, as indicated by gel chromatography and ion-exchange h.p.l.c. Rate-zonal centrifugation showed that the 'whole' mucins are polydisperse in size, with a weight-average Mr of (14-16) x 10(6). The macromolecules were observed by electron microscopy, as linear and apparently flexible thread-like structures. Subunits and T-domains had weight-average contour lengths of 490 nm and 160 nm respectively. It is concluded that mucus glycoproteins are present in secretions from the healthy lower respiratory tract. The 'whole' tracheal mucins are assembled from subunits, which in turn can be fragmented into high-Mr glycopeptides corresponding to the oligosaccharide domains typically found in mucus glycoproteins. The size and macromolecular architecture of the tracheal mucins is thus similar to that observed for mucins from human cervical mucus, chronic bronchitic sputum and pig stomach, providing yet another example of this general design of these macromolecules, i.e. subunits assembled end-to-end into very large linear and flexible macromolecules.     

New approaches to the problem of macronuclear assortment

Macronuclear assortment is a vegetative (somatic) process in the ciliate protozoan genus Tetrahymena which leads to the production of “homozygous” phenotypes from sexually-produced heterozygotes. The appearance of pure types follows a model of 45 randomly distributed and replicating subunits in the polyploid macronucleus. Models for the organization of macronuclear chromatin must explain the following: (1) haploid genomes are distributed at each fission; (2) not all loci assort; (3) different loci begin assortment at different times following conjugation. Therefore, either haploid subunits distributed at fission must frequently exchange parts (by somatic recombination, gene conversion, or progressive chromosome fragmentation), or they may actually disintegrate between fissions. Some of these possibilities can be distinguished by considering the kinetics of the appearance of pure types at two or more loci simultaneously. Conditions which give optimum discrimination between hypotheses of limited recombination and complete scrambling (by disintegration of haploid units between fissions) have been developed. A method for determining the number of assorting subunits in very young macronuclei is also given.     

Multi-scale calculation and global-fit analysis of hydrodynamic properties of biological macromolecules: determination of the overall conformation of antibody IgG molecules

We present a scheme, based on existing and newly developed computational tools, for the determination of the overall conformation of biological macromolecules composed by domains or subunits, using from such structural determination easily available solution properties. In a multi-scale approach, atomic-level structures are used to provide simple shapes for the subunits, which are put together in a coarse grained model, with a few parameters that determine the overall shape of the macromolecule. Computer programs, like those in the HYDRO suite that evaluate the properties of either atomic or coarse-grained models. In this paper we present a new scheme for a global fit of multiple properties, implemented in a new computer program, HYDROFIT, which interfaces with the programs of the HYDRO suite to find an optimum, best-fitting structure in a robust but simple way. The determination of the overall structure of the native antibody IgG3, bearing a long hinge, and that of the hingeless mutant m15 is presented to test and confirm the validity of this simple, systematic and efficient scheme.     

The crystal structure of the Methanocaldococcus jannaschii multifunctional L7Ae RNA-binding protein reveals an induced-fit interaction with the box C/D RNAs

Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that recognizes the K-turn motif in ribosomal, box H/ACA, and box C/D sRNAs. The crystal structure of Methanocaldococcus jannaschii L7Ae has been determined to 1.45 A, and L7Ae's amino acid composition, evolutionary conservation, functional characteristics, and structural details have been analyzed. Comparison of the L7Ae structure to those of a number of related proteins with diverse functions has revealed significant structural homology which suggests that this protein fold is an ancient RNA-binding motif. Notably, the free M. jannaschii L7Ae structure is essentially identical to that with RNA bound, suggesting that RNA binding occurs through an induced-fit interaction. Circular dichroism experiments show that box C/D and C'/D' RNA motifs undergo conformational changes when magnesium or the L7Ae protein is added, corroborating the induced-fit model for L7Ae-box C/D RNA interactions.     

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.     

Number of subunits comprising the epithelial sodium channel.

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type alphabeta-hENaC coexpressed with gamma-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one gamma subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.     

Statistical Model for Locating Fragile Points on Nucleic-ACID Molecules

Breakage is an important behavioral characteristic of nucleic-acid molecules. This paper concerns the problem of estimating the location of fragile (F) points on biological macromolecules such as DNA or RNA. We assume that the original length of the macromolecules is a random variable, breakage occurs more likely at some points of the macromolecules than others, and the number of breakages, except for those occurring at such points, is according to a Poisson process. Under these assumptions, the density function of the length of a randomly chosen segment has been derived. The Maximum Likelihood Estimation (MLE) is suggested for estimating the parameters. The model is applied to the data available on RNA of Avian Myeloblostosis Virus (AMV), and three possible F-points have been located. The robustness of the model is discussed.     

Solute size effects on the diffusion in biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

Dynamic model of protein behavior in water]

In the basis of the suggested model lies the hypothesis that during the evolution process biological macromolecules "learnt" to use the ability of water through cooperative transition from the rigid phase to the liquid one without a change in free energy for the regulation of their conformation. In accordance with the results of a number of investigations it is assumed that the protein molecule exists in thermodynamic equilibrium between two conformers with different accessibilities of nonpolar cavities to water and with different effective volumes. Deformation of both or one of the conformers under the effect of specific or nonspecific influences removes the system (protein+water) from the equilibrium, and as a result of the relaxation process the system achieves a new equilibrium state. A change in the equilibrium constant between the conformers determines the change of the average protein volume. The entropy and entalpy of protein and water in the system (protein+water) change during this process in a counterphase manner. Phase transition of water, involved between the subunits of oligomeric protein, may play a significant role in the mechanisms of allosteric effects.     

RNA structural motifs: building blocks of a modular biomolecule

RNAs are modular biomolecules, composed largely of conserved structural subunits, or motifs. These structural motifs comprise the secondary structure of RNA and are knit together via tertiary interactions into a compact, functional, three-dimensional structure and are to be distinguished from motifs defined by sequence or function. A relatively small number of structural motifs are found repeatedly in RNA hairpin and internal loops, and are observed to be composed of a limited number of common 'structural elements'. In addition to secondary and tertiary structure motifs, there are functional motifs specific for certain biological roles and binding motifs that serve to complex metals or other ligands. Research is continuing into the identification and classification of RNA structural motifs and is being initiated to predict motifs from sequence, to trace their phylogenetic relationships and to use them as building blocks in RNA engineering.     

OXPHOS susceptibility to oxidative modifications: the role of heart mitochondrial subcellular location

In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications.     

Stochastic analysis of a nonlinear model for selection of biological macromolecules

A stochastic analysis of a nonlinear selection model is presented. The model, based on Eigen and Schuster's theory of selection and evolution of biological macromolecules, considers the effects of fluctuations on the individual concentrations of macromolecules as well as the total population numbers in constrained systems. Our analysis shows that one of the models most often treated deterministically (referred to as constant organization in the literature) becomes unstable when fluctuations in the total population number are considered. An alternative model which apparently has built in self-regulating properties is analyzed and proves to be stable except for some special cases of degeneracy.     

Solute Size Effects on the Diffusion in Biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.