Fluorescence labels as sensors for oxygen binding of arthropod hemocyanins

The molecular basis of high cooperativity in multi-subunit proteins is still unknown in most cases. Oxygen binding by multi-subunit hemocyanins produces two intrinsic spectroscopic signals which are, however, either limited to the UV or are very weak. Here we demonstrate that fluorescence labels emitting in the visible can be used as sensors for cooperative oxygen binding of hemocyanins. Fluorescence resonance energy transfer to the oxygenated active sites quenches the emission of the labels by roughly 50% upon oxygenation of the protein. The labels give strong and photo-stable emission, allowing imaging of single hemocyanin molecules. Therefore, this study opens up a new perspective for investigating the molecular basis of cooperative oxygen binding at the single-molecule level. In addition, another novel application is provided by these labels, i.e., the investigation of the influence of effectors by recording simultaneously the binding of oxygen in the visible and of effectors in the UV.     

Flexibility, diversity, and cooperativity: pillars of enzyme catalysis

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, nuclear magnetic resonance, and single-molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model.     

Do biomolecules process information differently than synthetic organic molecules?

This paper compares information/signal processing in synthetic and biological molecules. The role of conformation-based (shape-based) mechanisms and electrostatic interactions in molecular recognition is discussed. In biological electron transfer, the 'electron shuttle'-mediated mechanism is contrasted with the mechanism based on pre-formed 'electron wires'. While biological information processing is thought to be more distributed (less discrete), an example of molecular switch is presented: visual transduction. We further speculate that visual transduction may be implemented in the form of a switch based on electrostatic interactions. The concept of intelligent materials is discussed with the well-known Bohr effect of hemoglobin oxygenation. Based on these examples, we argue that there are no fundamental differences between synthetic and biological molecules in their mode of information processing. In the pursuit of novel paradigms of molecular information processing, we also perceive no conflicts in developing molecular devices that emulate the switching function of conventional microelectronic devices.     

Evolutionary roots of genetic code

It is presented the modern state of the problem on abiogenic synthesis of molecular mechanisms connected with genetic information storage and transfer. On the base of experimental results it is suggested model of the common ancestor of tRNA and prime mechanism of the specific peptide synthesis.     

The role of structural reorganization in charge carrier transfer in a DNA molecule

The model proposed for hole transfer in DNA molecules with different configurations allows for the changes in the reorganization energy during charge transfer in a nucleotide strand with variations in the degree of orbital overlap in neighboring nucleotide pairs in different molecular sequences. The rate of hole transfer occurring in a DNA molecule through the superexchange and hopping transfer mechanisms is limited by the vibrational relaxation of the geometry of the nucleotide bases, as well as by the dynamics of solvent molecules. The rate of charge transfer in the DNA molecule depends on the height of the potential barrier between the donor fragment and the molecular bridge and on the positional arrangement of nucleobase pairs and their number in the molecular bridge. Inclusion of the interstrand charge transfer, which is characterized by a small degree of orbital overlap in the nucleobases of the opposite strands, does not affect the total charge transfer in the DNA molecule. An increase of the number of parallel components (processes) in the hopping mechanism entails an increase in the rate of charge transfer in the double helix.     

Coevolution of honest signaling and cooperative norms by cultural group selection

Evolution of cooperative norms is studied in a population where individual and group level selection are both in operation. Individuals play indirect reciprocity game within their group and follow second order norms. Individuals are norm-followers, and imitate their successful group mates. Aside from direct observation individuals can be informed about the previous actions and reputations by information transferred by others. A potential donor estimates the reputation of a potential receiver either by her own observation or by the opinion of the majority of others (indirect observation). Following a previous study (Scheuring, 2009) we assume that norms determine only the probabilities of actions, and mutants can differ in these probabilities. Similarly, we assume that individuals follow a stochastic information transfer strategy. The central question is whether cooperative norm and honest social information transfer can emerge in a population where initially only non-cooperative norms were present, and the transferred information was not sufficiently honest.It is shown that evolution can lead to a cooperative state where information transferred in a reliable manner, where generous cooperative strategies are dominant. This cooperative state emerges along a sharp transition of norms. We studied the characteristics of actions and strategies in this transition by classifying the stochastic norms, and found that a series of more and more judging strategies invade each other before the stabilization of the so-called generous judging strategy. Numerical experiments on the coevolution of social parameters (e.g. probability of direct observation and the number of indirect observers) reveal that it is advantageous to lean on indirect observation even if information transfer is much noisier than for direct observation, which is because to follow the majorities’ opinion suppresses information noise meaningfully.     

The effect of molecular inhibition on evolutionary learning: studies in the hypernetwork architecture

The hypernetwork architecture is a biologically inspired learning model based on abstract molecules and molecular interactions that exhibits functional and organizational correlation with biological systems. Hypernetwork organisms were trained, by molecular evolution, to solve N-input parity tasks. We found that learning improves when molecules exhibit inhibitory sites, allowing molecular inhibition and opening the possibility of forming negative feedback regulatory pathways. Optimal learning is achieved when at least 20% of the molecules in each cell have inhibitory sites. Intra-cellular as well as inter-cellular molecular inhibitions play an important role in the information processing of hypernetwork organisms, by maintaining a balance of the molecular cascade reactions. Similar mechanisms inside neurons are considered important for memory.     

Molecules and their migration in the universe

This paper outlines the assemblage of molecules observed in various astronomical objects found in, largely, the interstellar medium (not including the planets of the Solar System and their satellites). Various mechanisms for the spatial migration of molecules, including the transfer of molecular compounds from one planetary (protoplanetary) system to another, are considered. It is shown that over cosmological time the transfer of molecules is possible over distances up to 100 million light years. Hoyle and Wickramasinghe’s hypothesis on the biological origin of some interstellar dust grains is also briefly discussed.     

Semiotic Tools For Multilevel Cell Communication

Cell communication plays a key role in multicellular organisms. In developing embryos as in adult organisms, cells communicate by coordinating their differentiation through the establishment and/or renewal of a variety of cell communication channels. Under both these conditions, cells interact by either receptor signalling, surface recognition of specific cell adhesion molecules or transfer of cytoplasmic components through junctional coupling. In recent years, it has become apparent that cells may also communicate through the extracellular release of microvesicles. They may originate as either exosomes from the endosomal compartment upon fusion of multivesicular bodies with the plasma membrane or be shed directly from the plasma membrane via extensions of the cell surface. Microvesicles may disperse over long distances through body fluids and deliver their molecular cargo onto a variety of target cells. As a general rule, the metabolic fate of these cells is determined by the molecular nature of the vesicular cargo, while targeting itself depends on the affinity of the molecules expressed on the enclosing membrane. In this paper, we will be arguing that intercellular vesicular transfer is substantially different from other types of cell communication, allowing cells and molecules to interact on varying levels. Cells interacting via ligand signalling owe their specificity to the steric coupling with cognate receptor molecules. As such, it is a pure molecular process that affects target cells only upon integration into their responding repertoire. In this relationship, coupled cells are reciprocally adapted to each other through the selection of their respective signalling capacities, following exploration of their receptor specificity. Interaction by intercellular vesicles realizes a substantially different type of cell communication. Vesicular traffic allows donor cells to carry out a horizontal type of gene transfer and target this information over long distances via independently controlled mechanisms. Because of this independence, cells interacting via vesicular traffic are not expected to adapt their signalling correspondences, but to control instead the efficiency of their cargo delivery irrespective of the receptor repertoire expressed by the target tissue. In this paper, the multifaceted functions of the intercellular vesicular traffic will be discussed in a multilevel biosemiotic perspective with the aim of unravelling the cellular mechanisms devised by nature to accomplish communication.     

Model building of a protein-protein complexed structure using saturation transfer and residual dipolar coupling without paired intermolecular NOE

For understanding the precise mechanisms of molecular recognition of proteins, three-dimensional structural analyses of the protein-protein complexes are essential. For this purpose, a new method to reveal complex structures was developed with the assistance of saturation transfer (SAT) and residual dipolar coupling (RDC) by heteronuclear NMR experiments, without any paired intermolecular NOE information. The SAT and RDC experiments provide the information of the interfacial residues and the relative orientations of the two protein molecules, respectively. Docking simulation was then made to reconstruct a complex conformation, which satisfies the SAT and RDC data. The method was applied to the CAD-ICAD complex structure, which was previously determined by the NOE-distance geometry method. The quality of the current model was evaluated.     

Exosomes: vesicular carriers for intercellular communication in neurodegenerative disorders

The intercellular transfer of misfolded proteins has received increasing attention in various neurodegenerative diseases characterized by the aggregation of specific proteins, as observed in Alzheimer’s, Parkinson’s and Huntington’s disease. One hypothesis holds that intercellular dissemination of these aggregates within the central nervous system results in the seeded assembly of the cognate soluble protein in target cells, similar to that proposed for transmissible prion diseases. The molecular mechanisms underlying the intercellular transfer of these proteinaceous aggregates are poorly understood. Various transfer modes of misfolded proteins including continuous cell-cell contacts such as nanotubes, unconventional secretion or microvesicle/exosome-associated dissemination have been suggested. Cells can release proteins, lipids and nucleic acids by vesicular exocytosis pathways destined for horizontal transfer. Encapsulation into microvesicular/exosomal vehicles not only protects these molecules from degradation and dilution in the extracellular space but also facilitates delivery over large distances, e.g. within the blood flow or interstitial fluid. Specific surface ligands might allow the highly efficient and targeted uptake of these vesicles by recipient cells. In this review, we focus on the cell biology and function of neuronal microvesicles/exosomes and discuss the evidence for pathogenic intercellular protein transfer mediated by vesicular carriers.     

The role of structural reorganization in charge carrier transfer in DNA molecule

A model of hole transfer in DNA molecules has been proposed, which takes into account changes in the reorganization energy and orbital coupling between the neighboring bases during the charge transfer in different molecular sequences. It is shown that the rate of hole transfer by the superexchange and hopping transfer mechanisms is limited by the relaxation of the geometries of nucleobases participating in charge migration and the dynamics of solvent molecules. The rate of charge transfer in the DNA molecule is found to be dependent on the height of the potential barriers between the nucleotide and the molecular sequences. The inclusion of the interchain charge transfer, which is characterized by weak coupling between the nucleotides located in opposite strands, does not affect the general charge transport in DNA. The increase in the number of the parallel components of the hopping mechanism leads to a rise in the charge transfer rate in the double helix.     

Transfer factor in the age of molecular biology: A review

Current data suggests that the transferring of immunologically specific information by transfer factor molecules requires interaction with a cell that has been genetically programmed to be antigen reactive but at the time of interaction is unprimed. Contact with transfer factor molecules would allow a naive recipient, on a first encounter with antigen, to make a secondary rather than a primary immunological response. Transfer factor molecules for each and every antigenic determinant are thus necessary. Transfer factors made from animals or humans are capable of transferring antigen specificity across a species barrier. Even primitive species have cells from which one can make transfer factors. The molecules are, therefore, well conserved and it is reasonable to suggest that they are important for normal immunological functioning. Proposed mechanisms of action must explain the fact that transfer factors obtained from the cells of high responder animals are capable of transferring delayed hypersensitivity to low responder animals while the reverse is not true. Transfer factor molecules are likely to interact with the variable regions of the alpha and/or beta chain of T cell receptors to change their avidity and affinity for antigen in a way that otherwise would only occur after an encounter with antigen.     

Neurochemical aspects of interneuronal communication

It is suggested that the term neurotransmission, which is used to designate neuronal communication at synaptic level, be associated to the less restrictive term neuromodulation. These two types of intercellular communication seem in fact to be two basically different mechanisms, both of which contribute to neuronal integration. The integration of neuronal information at cellular level appears to be more complex than the simple addition of excitatory plus inhibitory influences eliciting postsynaptic responses. Evidence has been obtained that non synaptic transmission can alter the capacity of a given synapse to transfer neuronal information from the presynaptic element to the postsynaptic neuron. For instance, presynaptic mechanisms provide evidence for the functional independence of the nerve terminals, since the release of neuromediators by the latter is sometimes independent of the axonal firing rate. Similarly, the somato-dendritic part of some neurons exhibits intrinsic functions, such as a dendritic release of neuromediator, suggesting that the control of the axonal firing rate takes place partly at this somato-dendritic level and does not depend for the totality on afferent axonic information. The intercellular operations which organize individual neurons into neuronal networks will also occur either at somato-dendritic level or at the level of specific nerve terminals selected as the result of presynaptic interactions. This integration of neuronal information also seems to take place at postsynaptic level, where cooperative interactions have been shown to occur between various receptors. These mechanisms will function at the level of a single nerve terminal containing more than one neuromediator. Neuromodulation can therefore be said to involve very efficient adaptive processes, which help to account for the fact that such large behavioral responses are expressed by such a small number of neuronal elements.     

Molecules as electronic components?

Aspects of electronics at the molecular level are reviewed. Molecules can store information in their different states. To transmit information, they need to interact with other molecules, but this affects the states. Excitation transfer is also more complex than sometimes realised. Information processing is best treated in terms of the whole system of interacting molecules, so that molecules cannot be treated simply as small conventional electronic components.     

Advances in defining signaling networks for the establishment of neuronal polarity

Neurons are highly polarized cells that have structurally and functionally distinct processes called axons and dendrites. How neurons establish polarity is one of the fundamental questions of neuroscience. In the last decade, significant progress has been made in identifying and understanding the molecular mechanisms responsible for neuronal polarization, primarily through researches conducted on cultured neurons. Advances in phosphoproteomics technologies and molecular tools have enabled comprehensive signal analysis and visualization and manipulation of signaling molecules for analyzing neuronal polarity. Furthermore, advances in gene transfer techniques have revealed the role of extracellular and intracellular signaling molecules in neuronal polarization in vivo. This review discusses the latest insights and techniques for the elucidation of the molecular mechanisms that control neuronal polarity.     

NMR spectroscopic and molecular modeling studies of protein-carbohydrate and protein-peptide interactions

Investigations of the conformations of carbohydrates, their analogues and their molecular mimics are described, with emphasis on structural and functional information that can be gained by NMR spectroscopic techniques in combination with molecular modeling. The transferred nuclear Overhauser effect (trNOE) has been employed to determine the bound conformations of carbohydrates and other bioactive molecules in complex with protein receptors. The corresponding experiments in the rotating frame (trROE) and selective editing experiments (e.g., QUIET-NOESY) are used to eliminate indirect cross-relaxation pathways (spin diffusion), thereby minimizing errors in the data used for calculation of conformations. Saturation transfer difference NMR experiments reveal detailed information about intermolecular contacts between ligand and protein. Computational techniques are integrated with NMR-derived information to construct structural models of these bioactive molecules and of their complexes with proteins. Recent investigations into the nature of molecular mimicry with regard to protein-ligand interactions are described, along with applications in determining the mode of action of enzyme inhibitors. The results are relevant for the design of the next generation of drug and vaccine candidates.     

Posttransition state desolvation of the hydrophobic core of the src-SH3 protein domain

The folding thermodynamics of the src-SH3 protein domain were characterized under refolding conditions through biased fully atomic molecular dynamics simulations with explicit solvent. The calculated free energy surfaces along several reaction coordinates revealed two barriers. The first, larger barrier was identified as the transition state barrier for folding, associated with the formation of the first hydrophobic sheet of the protein. phi values calculated from structures residing at the transition state barrier agree well with experimental phi values. The microscopic information obtained from our simulations allowed us to unambiguously assign intermediate phi values as the result of multiple folding pathways. The second, smaller barrier occurs later in the folding process and is associated with the cooperative expulsion of water molecules between the hydrophobic sheets of the protein. This posttransition state desolvation barrier cannot be observed through traditional folding experiments, but is found to be critical to the correct packing of the hydrophobic core in the final stages of folding. Hydrogen exchange and NMR experiments are suggested to probe this barrier.     

Positive cooperativity in the functioning of molecular chaperone GroEL.

In the presence of its partner, GroES, the tetradecameric molecular chaperone GroEL binds 14 ATP molecules, half of which are hydrolyzed in a cooperative manner. Moreover GroEL can bind, with a positive cooperativity, more than two molecules of nonfolded protein rhodanese. The role of the cooperative mechanism in the functioning of GroEL is discussed.     

GRIPDB - G protein coupled Receptor Interaction Partners DataBase

The G protein Coupled Receptor (GPCR) superfamily is one of the most important pharmaceutical targets. Studies of GPCRs have long been performed under the assumption that GPCRs function as monomers. However, recent studies have revealed that many GPCRs function as homo- and/or hetero-dimers or higher-order oligomeric molecular complexes. As a result, information about GPCR oligomerization is rapidly accumulating, although the molecular mechanisms of oligomerization are not fully understood. A comprehensive collection of information about oligomerization would accelerate investigations of the molecular mechanisms of GPCRs' oligomerization and involvement in signaling. Hence, we have developed a database, G protein coupled Receptor Interaction Partners DataBase (GRIPDB), which provides information about GPCR oligomerization. The entries in the database are divided into two sections: (I) Experiment Information section and (II) Prediction Information section. The Experiment Information section contains (I-i) experimentally indentified GPCR oligomers and their annotations, and (I-ii) experimentally suggested interfaces for the oligomerization. Since the number of experimentally suggested interfaces is limited, the entries in the Prediction Information section have been introduced to provide information about the oligomerization interfaces predicted by our computational method. The experimentally suggested or computationally predicted interfaces are displayed by 3D graphics, using GPCRs with available coordinates. The information in the GRIPDB, especially that about the interfaces, is useful to investigate the molecular mechanisms of signal transduction via GPCR oligomerization. The GRIPDB is available on the web at the following URL: http://grip.cbrc.jp/GDB/index.html .