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The cellular interior is composed of a variety of microenvironments defined by distinct local compositions and composition-dependent intermolecular interactions. We review the various types of nonspecific interactions between proteins and between proteins and other macromolecules and supramolecular structures that influence the state of association and functional properties of a given protein existing within a particular microenvironment at a particular point in time. The present state of knowledge is summarized, and suggestions for fruitful directions of research are offered.

Biology and biochemistry students must learn to visualize and comprehend the complex three-dimensional (3D) structures of macromolecules such as proteins or DNA. However, most tools available for teaching biomolecular structures typically operate in two dimensions. Here, we present protocols and pedagogical approaches for using immersive augmented reality (AR) visors, specifically the Microsoft HoloLens, to reinforce learning with large scale 3D holographic structures. We developed a novel workflow to render vividly colored custom biomolecules in AR visors. In addition, we developed AR exercises to review concepts relevant to protein or DNA structure and then implemented the exercises in four different biology and biochemistry courses. Surveys showed that students reported greater interest in biomolecular structures after the exercise. We also highlight some of the advantages and disadvantages of the software and hardware of this upcoming technology., (© 2020 International Union of Biochemistry and Molecular Biology.)

In vitro assays have contributed important insights into the mechanisms of RNA metabolism in cells. A growing collection of microscopy techniques is allowing the measurement of macromolecular binding and complex formation in the context of a real cell. We will first discuss two of the more established techniques. Fluorescence resonance energy transfer (FRET) identifies binding partners, pairs of molecules residing in the same macromolecular complexes. The complimentary technique of fluorescence recovery after photobleaching (FRAP) measures the rates of binding and unbinding of those molecules in their complexes. A newer technique--in vitro FRAP--assesses the regulation of binding and complex formation by co-factors in the nucleus.

To understand the cell, we need to determine the macromolecular assembly structures, which may consist of tens to hundreds of components. First, we review the varied experimental data that characterize the assemblies at several levels of resolution. We then describe computational methods for generating the structures using these data. To maximize completeness, resolution, accuracy, precision, and efficiency of the structure determination, a computational approach is required that uses spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. This approach is illustrated by determining the configuration of the 456 proteins in the nuclear pore complex (NPC) from baker's yeast. With these tools, we are poised to integrate structural information gathered at multiple levels of the biological hierarchy--from atoms to cells--into a common framework.

An improved method to analyze the (13)C NMR spectra of native starches, which considers the contribution of the V-type conformation and the nature of the amorphous component, has been developed. Starch spectra are separated into amorphous and ordered subspectra, using intensity at 84 ppm as a reference point. The ordered subspectra of high amylose starches show the presence of both V-type single helices and B-type double helices. Relative proportions of amorphous, single, and double-helical conformations are estimated by apportioning intensity of C1 peak areas between conformational types on the basis of ordered and amorphous subspectra of the native starch. Quantitative analysis shows that the V-type single-helical component increases with amylose content of starches. Different amorphous subspectra are needed to provide a consistent analysis of granular starches from diverse sources. The method of preparation was found to be more important than the starch botanical origin in determining (13)C NMR spectral features of amorphous samples.

The composition and rheological properties of beta-lactoglobulin/pectin coacervates have shown significant correlations with sodium chloride concentration (C(NaCl)) and initial protein/polysaccharide ratio (r). An increase of C(NaCl) from 0.01 to 0.21 M at r = 5:1 leads to the increase in both beta-lactoglobulin and pectin contents in the coacervates, which can be explained in terms of salt-enhanced effect at lower salt concentrations. Further increase of C(NaCl) from 0.21 to 0.41 M decreases the proportions of these two biopolymers in the coacervates, exhibiting salt-reduced effect at higher salt concentrations. Moreover, the stronger self-aggregation of beta-lactoglobulin with increasing salt concentration gives rise to a decreasing actual protein/polysaccharide ratio in the coacervates at 0.01-0.21 M C(NaCl) and r = 5:1. An increase of r from 5:1 to 40:1 often increases the actual amount of pectin chains in beta-lactoglobulin/pectin coacervates, but it exhibits a maximum in beta-lactoglobulin content at r = 20:1. A much higher storage modulus (G') than loss modulus (G' ') for all beta-lactoglobulin/pectin coacervates suggests the formation of highly interconnected gel-like structure. The values of G' increase as C(NaCl) increases from 0.01 to 0.21 M, whereas a further increase of C(NaCl) from 0.21 to 0.41 M causes G' values to decrease to much lower values. These results further disclose the salt-enhanced effect and the salt-reduced effect at low and high salt concentrations, respectively. On the other hand, increasing r from 5:1 to 40:1 favors the formation of stronger gel-like beta-lactoglobulin/pectin coacervates, which mainly originates from the higher actual amount of pectin chains in beta-lactoglobulin/pectin coacervates at higher r values.

Keratins are self-organized proteins that are abundantly available in wool, feather, human hair, etc., making them a potential cheap feedstock for the modification of amino acids. This paper explores the hydrolysis of keratin in water under specific pressure-temperature conditions where the hydrolysis through scission of the protein chain yields oligopeptides. Here we report for the first time that, under appropriate conditions, these oligopeptides self-assemble into a hierarchical architecture, the process being followed in time by optical microscopy. Birefringent needle-like crystals are observed which tend to nucleate heterogeneously. When given sufficient time, these needles become tens of microns in length and act as further nuclei, developing a highly repetitive structure of several hundreds of microns in size. Micro-focus X-ray diffraction studies supported by in situ microscopy reveal that these needles have a crystal structure similar to that of the native protein, although better organized along the ab-plane. Spectroscopic studies on these structures show crystalline bands that disappear above 150 degrees C, coinciding with an endothermic peak in DSC. Amino acid analysis shows that the self-assembled birefringent entities are indeed oligopeptides, consisting of sequences of approximately 40 amino acids. The proposed ecofriendly route provides an effective route for obtaining oligopeptides that can be used as important building blocks for the synthesis of a range of novel polymers. The oligopeptides obtained from the sustainable source can be used as important building blocks for the synthesis of a range of novel polymers.

A variety of organocatalysts has been surveyed in the ring opening polymerization of trimethylene carbonate. Excellent control was found for several of these catalysts yielding well-defined polycarbonates with molecular weights up to 50,000 g mol(-1), polydispersities below 1.08, and high end-group fidelity. Melt or bulk polymerization was accomplished without loss of control of molecular weight or polydispersity, and random ester-carbonate bulk polymerizations were also demonstrated. Furthermore, by combining disparate polymerization techniques using bifunctional initiators, the mild polymerization conditions allow for the preparation of new block copolymers. Hydrogen-bond activation of monomer and initiator/propagating species is proposed as the underlying mechanism, which can be tuned to mitigate adverse side reactions.

Mammalian gastric mucin, at high concentration, is known to form a gel at low pH, behavior essential to the protection of the stomach from auto-digestion. Atomic force microscopy (AFM) measurements of dilute solutions of porcine gastric mucin in an aqueous environment in the pH range 6-2 provide a direct visualization of extended fiberlike molecules at pH 6 that aggregate at pH 4 and below forming well-defined clusters at pH 2. The clusters consist of 10 or less molecules. AFM images of mucin at high concentration at pH 2 reveal clusters similar to those seen in the dilute solutions at low pH. We also imaged human gastric mucus revealing a network having a "pearl necklace" structure. The "pearls" are similar in size to the clusters found in the purified porcine gastric mucin gels. AFM images of deglycosylated mucin reveal that the deglycosylated portions of the molecule re-fold into compact, globular structures suggesting that the oligosaccharide chains are important in maintaining the extended conformation of mucin. However, the oligosaccharides do not appear to be directly involved in the aggregation at low pH, as clusters of similar size are observed at pH 2 in both native and deglycosylated mucin.

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.

Reactivity data related to processes in which molecular receptors promote the reaction of two simultaneously complexed reactants have been surveyed and analyzed in terms of effective molarity (EM). Methods and criteria for the calculation of reliable EM's have been highlighted. Extension of a previous extrathermodynamic treatment of intramolecular reactions of bifunctional chains to the intracomplex reactions of the ternary complexes involved in two-substrate catalyzed reactions has provided a sound framework for a comparative analysis of reactivity and catalytic efficiency in structurally diverse and apparently unrelated systems.

Rational and efficient process development in chemical technology always makes heavy use of process analysis in terms of balances, kinetics, and thermodynamics. While the first two of these concepts have been extensively used in biotechnology, it appears that thermodynamics has received relatively little attention from biotechnologists. This state of affairs is one among several reasons why development and design of biotechnological processes is today mostly carried out in an essentially empirical fashion and why bioprocesses are often not as thoroughly optimized as many chemical processes. Since quite a large body of knowledge in the area of bio thermodynamics already existed in the early nineties, the Steering Committee of a European Science Foundation program on Process Integration in Biochemical Engineering identified a need to stimulate a more systematic use of thermodynamics in the area. To this effect, a bianual course for advanced graduate students and researchers was developed. The present contribution uses the course structure to provide an outline of the area and to characterize very briefly the achievements, the challenges, and the research needs in the various sub-topics.

A method is presented for producing a seed-stock mixture for macromolecular crystallization. A PTFE bead and micro-centrifuge tube act as mortar and pestle for pulverizing seed crystals of macromolecules. Energy for the bead's motion is supplied by a vortex mixer or an ultrasonic bath. The crushed crystal is serially diluted to prepare a seed-stock mixture of the desired concentration for crystallization. Crystals produced using both hanging-drop vapor diffusion and a capillary microbatch method show expected dilution behavior. This technique of producing seed stock is compared with traditional means and advantages over the standard protocol are demonstrated.

The fixed-point algebraic method [Storer and Cornish-Bowden (1976) Biochem. J. 159, 1-5] for computing the concentrations at equilibrium of complex biochemical mixtures fails for many binding stoichiometries, especially those that include molecular self-association. A typical example is the monomer-dimer-tetramer equilibrium. This paper reports two main results. First, the above algorithm is analysed theoretically to predict for which binding stoichiometries it succeeds and for which it will fail. Secondly, an alternative algorithm is described for self-associating biochemical systems. Illustrative examples are based on the dimeric proteinase from HIV.

The success of the original yeast two-hybrid system has stimulated the development of a number of 'hybrid technologies' in yeast (and now prokaryotes and mammals) to widen the scope of the protein-protein interactions that can be analyzed, and to enable comparable studies of the interactions of proteins with DNA, RNA or small chemical ligands. In addition, the application of the two-hybrid system to entire genomes is being used to create protein linkage maps which catalog the network of interactions of an organism's complete proteome.

Training newcomers to the field of macromolecular modeling is as difficult as is training beginners in x-ray crystallography, nuclear magnetic resonance, or other methods in structural biology. In one or two lectures, the most that can be conveyed is a general sense of the relationship between modeling and other structural methods. If a full semester is available, then students can be taught how molecular structures are built, manipulated, refined, and analyzed on a computer. Here we describe a one-semester modeling course that combines lectures, discussions, and a laboratory using a commercial modeling package. In the laboratory, students carry out prescribed exercises that are coordinated to the lectures, and they complete a term project on a modeling problem of their choice. The goal is to give students an understanding of what kinds of problems can be attacked by molecular modeling methods and which problems are beyond the current capabilities of those methods.

A repeating particle associated with the cristae and the inner membrane of the external envelope has been recognized and characterized in beef heart mitochondria by correlated electron microscopic and biochemical studies. Many thousands (ca. 10(4) to 10(5)) of these particles, disposed in regular arrays, are present in a single mitochondrion. The repeating particle, called the elementary particle (EP), consists of three parts: (1) a spherical or polyhedral head piece (80 to 100 A in diameter); (2) a cylindrical stalk (about 50 A long and 30 to 40 A wide); and (3) a base piece (40 x 110 A). The base pieces of the elementary particles form an integral part of the outer dense layers of the cristae. The elementary particles can be seen in electron micrographs of mitochondria in situ, of isolated mitochondria, and of submitochondrial particles with a complete electron transfer chain. Negative staining with phosphotungstate is only one of several techniques that can be used for reproducible demonstration of the repeating particles and underlying subunit organization of mitochondrial membranes. A particulate unit containing a complete electron transfer chain can be isolated from beef heart mitochondria. The isolated unit approximates in size that of the elementary particle in situ. The molecular weight of the particle in situ is calculated to be 1.3 x 10(6). Evidence is presented for identifying the isolated unit with the elementary particle visualized in situ. The elementary particle of the mitochondrion is believed to be a prototype of a class of functional particles or macromolecular assemblies of similar size found in association with membranes generally.

Kinetic and ultracentrifugal experiments demonstrated that the previously described subcomponents of human C'1, designated C'1q, C'1r, and C'1s, interacted with each other in liquid phase to form a macromolecule which was then capable of converting sensitized erythrocytes (EA) to the state EAC'1. The apparent sedimentation constants of C'1q, C'1r, and C'1s and of the macromolecular product of their interaction were approximately 11S, 7S, 4S, and 18S respectively. Association of C'1 subcomponents was prevented and dissociation of macromolecular C'1 was effected by Na(3)HEDTA and Na(2)MgEDTA but not by Na(2)CaEDTA. The rate of formation of macromolecular C'1 was a function of concentration of subcomponents and temperature of interaction, with an apparent energy of activation of 21,000 calories per mol. Ultracentrifugal studies further indicated the macromolecular nature of C'1 in normal human serum. In the absence of EDTA, C'1 sedimented with the serum macroglobulins and C'1 subcomponents were not detected. Conversely, in the presence of EDTA, macromolecular C'1 was not demonstrable and individual C'1 subcomponents could be measured in lighter fractions. The significance of these observations in relation to previous studies on C'1 subcomponents, the role of Ca(++) in C'1 function, and the subunit structure of Enzymes has been discussed.

An allosteric model of an open monosubstrate enzyme reaction is analyzed for the case where the enzyme, containing two protomers, is activated by the product. It is shown that this system can lead to instabilities beyond which a new state organized in time or in space (dissipative structure) can be reached. The conditions for both types of instabilities are presented and the occurrence of a temporal structure, consisting of a limit cycle behavior, is determined numerically as a function of the important parameters involved in the system. Sustained oscillations in the product and substrate concentrations are shown to occur for acceptable values of the allosteric and kinetic constants; moreover, they seem to be favored by substrate activation. The model is applied to phosphofructokinase, which is the enzyme chiefly responsible for glycolytic oscillations and which presents the same pattern of regulation as the allosteric enzyme appearing in the model. A qualitative and quantitative agreement is obtained with the experimental observations concerning glycolytic self-oscillations.