Search

We present a detailed heuristic method to quantify the degree of local energetic frustration manifested by protein molecules. Current applications are realized in computational experiments where a protein structure is visualized highlighting the energetic conflicts or the concordance of the local interactions in that structure. Minimally frustrated linkages highlight the stable folding core of the molecule. Sites of high local frustration, in contrast, often indicate functionally relevant regions such as binding, active, or allosteric sites.

Digital proximity ligation assay (PLA) detects single protein molecules with a pair of oligonucleotide-conjugated antibodies and digital PCR (dPCR) readout, which allows absolute quantitation of proteins in single cells with high sensitivity. The pipeline also allows simultaneous measurement of protein and mRNA from the same single cell. The sensitivity of the assay has been further improved with implementation of the assay on a microfluidic system, which enables quantitation of rare protein species, with expression level as low as ~3000 protein molecules per cell.

Gelation is an efficient way to fabricate fibrous protein materials. Briefly, it is an aggregation process where protein molecules assembly from a random structure into an organized structure such as nanofibrillar networks. According to their mechanisms, the fibrous proteins gelation can be classified into physical gelation and chemical gelation. The physical gelation is formed by the conformational transformation of fibroin proteins, which can be triggered by temperature, concentration, pH, or shear force. On the other hand, the chemical gelation is to cross-link fibrous proteins through chemical and/or enzymatic reactions. In this chapter, we summarize the protocols for preparing fibrous protein hydrogels, including both physical and chemical methods. The mechanisms of these gelation methods are also highlighted.

Memdock is a tool for docking α-helical membrane proteins which takes into consideration the lipid bilayer environment. Given two α-helical membrane located protein molecules, the method outputs a list of potential complexes sorted by energy criteria. The program includes three steps: docking, refinement, and re-ranking of the results. All three docking steps have been customized to the membrane environment in order to improve performance and reduce program run-time. In this chapter, we describe the application of our web server, referred to as Memdock, for prediction of the docking complex for a pair of input membrane protein structures. Memdock is freely available for academic users without registration at http://bioinfo3d.cs.tau.ac.il/Memdock/index.html .

RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq.

Nanopore technology enables the detection and analysis of single protein molecules. The technique measures the ionic current passing through a single pore inserted in an electrically insulating membrane. The translocation of the protein molecule through the pore causes a modulation of the ionic current. Analysis of the ionic current reveals the biophysics of co-translocational unfolding and may be used to infer the amino acid sequence and posttranslational modifications of the molecule.

VNAR domains are the binding regions of new antigen receptor proteins (IgNAR) which are unique to sharks, skates, and rays (Elasmobranchii). Individual VNAR domains can bind antigens independently and are the smallest reported adaptive immune recognition entities in the vertebrate kingdom. Sharing limited sequence homology with human immunoglobulin domains, their development and use as biotherapeutic agents require that they be humanized to minimize their potential immunogenicity. Efforts to humanize a human serum albumin (HSA)-specific VNAR, E06, resulted in protein molecules that initially had undesirable biophysical properties or reduced affinity for cognate antigen. Two lead humanized anti-HSA clones, v1.10 and v2.4, were subjected to a process of random mutagenesis using error-prone PCR. The mutated sequences for each humanized VNAR variant were screened for improvements in affinity for HSA and biophysical properties, achieved without a predicted increase in overall immunogenicity.

Protein nanotechnology research is at the intersection of protein biology and nanotechnology. Protein molecules are repurposed as nanostructures and nanoscaffolds, and nanoscale tools are used to investigate protein assembly and function. In this chapter, a select review is given of some of the recent examples of protein nanostructures, covering both those directly borrowed from biology and those designed for use in nanotechnology. It updates the introductory chapter to Edition 2 of this volume to reflect significant progress in this field. Some strategies to incorporate protein structures into devices are also covered, with the successes and challenges of this interdisciplinary field identified. This provides an overarching framework for the rest of the volume, which details the case studies of some of the protein building blocks that have been designed and produced, along with tips and tools for their incorporation into devices and making functional measurements.

Protein drugs have revolutionized the pharmaceutical industry, offering new treatments for serious diseases. Since the first recombinant protein drug, Eli Lilly’s Humulin®, was approved 30 years ago (1), protein drugs have grown from esoteric specialty products to a major drug class. Of the 20 top selling drugs in the USA in the third quarter of 2012, nine are proteins. Despite these successes, the inherent instability of protein molecules remains an impediment to their development and to their safety and efficacy. One of the most serious types of instability is aggregation, the self-association of native protein through covalent and/or non-covalent interactions. Protein aggregates have been associated with increased or decreased drug potency and with an increased potential for immunogenic side effects, which can be life-threatening. In this themed issue of the AAPS Journal, we have assembled research and review articles that address the aggregation of therapeutic proteins. The issue was inspired by presentations at the 10th Annual Garnet E. Peck Symposium in Industrial Pharmacy, held at Purdue University in West Lafayette, Indiana, on October 11, 2012. At a practical level, interest in the aggregation of therapeutic proteins is driven by the need for stable formulations and robust manufacturing conditions. At the symposium, Dr. David Volkin (Dept. of Pharmaceutical Chemistry, University of Kansas) presented a series of case studies based on his recent work that address these issues. The cases showed the effects of excipients on the aggregation of an albumin fusion protein (2) and IgG2 monoclonal antibodies (mAbs) (3), the role of infusion bags in the solubility and aggregation of IgG4 mAbs (4), methods to ensure comparability during mAb process development (5), and an empirical phase diagram approach to identifying formulations that inhibit aggregation (6). In their review article in this special issue, Dr. Volkin and his coauthors summarize these case studies, present an overview of protein aggregation mechanisms, and describe high throughput approaches to monitoring protein stability (7). In developing drug products, the industry makes use of accelerated stability studies to estimate shelf life at the storage temperature based on degradation rates measured at higher temperatures. This extrapolation usually assumes that the temperature dependence of reaction rates follows Arrhenius behavior. However, protein aggregation often exhibits non-Arrhenius temperature dependence, even over relatively narrow temperature ranges. At the symposium, Dr. Chris Roberts (Dept. of Chemical and Biomolecular Engineering, University of Delaware) presented his group’s recent work on the mechanisms of protein aggregation and origins of non-Arrhenius behavior (8,9). In this themed issue, he and Dr. Wei Wang (Pfizer BioTherapeutics) summarize these mechanistic insights and discuss the implications for accelerated stability testing (10). Ensuring that protein drugs are aggregate-free requires robust, reproducible analytical methods. The ideal method would resolve aggregate and monomeric species, quantify aggregate size and concentration, and provide a low limit of detection, all while achieving high throughput at moderate cost. Current methods fall short of this ideal. For example, size exclusion chromatography (SEC) assays for soluble aggregates are expensive and low-throughput. Gel electrophoresis (e.g., SDS-PAGE) gives somewhat higher throughput but generally is not quantitative. To address these limitations, Dr. Mary Wirth (Dept. of Chemistry, Purdue University) and her colleagues are developing novel chromatographic materials based on silica colloidal crystals. These ordered arrays of silica particles provide plate heights in the low nanometer range, allowing for high resolution and rapid analysis times. For example, using a capillary packed with silica colloidal crystals and pressure-driven flow, the group has separated a monoclonal antibody and its aggregates in less than a minute with baseline resolution (11). Dr. Wirth and her coauthors summarize the group’s recent results in this themed issue (12). Together, these articles provide insight into our current understanding of protein aggregation. Gaps in fundamental understanding and applied technology remain, however, impeding our ability to monitor and inhibit aggregation in biologics. These gaps include limited understanding of the relative importance of partial unfolding, colloidal interactions, and chemical reaction in the protein aggregation process; a lack of understanding of the chemical and physical determinants of the immune response to protein drugs and their aggregated forms; poor agreement among available analytical methods for determining aggregate size and concentration, particularly in the subvisible range; and an incomplete understanding of the mechanistic effects of process stresses and formulation variables. We hope that the practical and theoretical perspectives assembled here will stimulate additional discussion and research in this important area.

Assume a sample of a fairly dilute protein solution, 1nM ( = 50ng∕ml if M r = 50kDa). Then 1μl will contain 1fmol of protein. Multiplied with Avogadro’s number (6. 022e23mol−1) this still corresponds to 6e8 protein molecules.

Our current knowledge about biological membranes shows that they belong to the most important cell structures. Mass transport and signal transduction obviously appear to be vital to physiological functions of biomembranes as they enable cellular compartmentalization and control over it at the same time. Many experiments and theoretical considerations in the past decades have shown that membranes consist of a laterally heterogeneous lipid bilayer with a large number of different protein molecules embedded in the lipid bilayer. Heterogeneity exists at any level — from the biochemical to the physical level, meaning that different constituents and supramolecular structures in membranes interact via different interactions and exhibit different motional characteristics. This complexity — as the most striking property of any biological system — remains a tough problem also for the up-to-date experimental and theoretical approaches.

Human leukocyte antigen (HLA) recognizes antigenic fragments and presents them to T cells. HLA is polymorphic. There are over 2000 different HLA alleles at present and the number is constantly increasing. However, antigen binding studies are limited to a small proportion of these alleles; the binding specificities of most alleles are unknown. Several research groups have attempted to partition different HLA alleles into groups. In this chapter previous classifications are reviewed and we present two chemometric approaches to classifying class I HLA alleles. The program GRID is used to calculate interaction energy between protein molecules and defined chemical probes. These interaction energy values are imported into another program GOLPE and used for principal component analysis (PCA) calculation, which groups HLA alleles into supertypes. Amino acids that are involved in the classification are displayed in the loading plots of the PCA model. Another method, hierarchical clustering based on comparative molecular similarity indices (CoMSIA) is also applied to classify HLA alleles and the results are compared with those of the PCA models.

The interest in trace elements in haemodialysed (HD) patients has been generated by the recognition that Al accumulation may cause toxicity. Anomalies of other trace elements in specific tissues are however commonly presents in HD patients (Gallieni, Brancaccio and Cozzolino, 1996) although the clinical relevance of these abnormalities remains unclear. Recent attention has been devoted to elevated level of Chromium in tissues of HD patients (Wallaeys et al., 1986) in contrast with very low essential levels in normal subjects (Minoia et al., 1990). A study by Wallaeys et al. described the pathways involved in transport of Cr across the dialysis membrane (Wallaeys, Cornelis, Sabbioni, 1988). Padovese et al. confirmed that fresh peritoneal dialysate contained a Cr level 17 fold above the indicative reference plasma Cr concentration (Padovese et al., 1992). Borquet et al. studied the kinetics of Cr in Continuous Ambulatory Peritoneal Dialysis patients (Borguet et al., 1996) and justified, with mathematical models, Cr accumulation in blood and tissues. In haemodiafiltration (HDF), the reinfusion fluids and large membrane pores, which allow the passage of larger protein molecules, are additional factors influencing the balance of trace elements (Van Renterghem, Cornelis and Vanholder, 1992). Accumulation or depletion of trace elements may be more relevant if we use large quantities of reinfusate, as in the case of on-line production of reinfusate (Canaud et al., 1998). For these reasons we studied Cr levels in patients treated by HDF with large amounts of on-line produced reinfusate and dialysate. We compared Cr whole blood concentrations with those of our HD patients and studied Cr concentrations in on-line HDF patients through an extended period. In order to guarantee safety of HDF patients, other trace elements, as well as Cr, were monitored both in blood and in dialysis fluids.

Proteins often form crystals of different shapes and sizes, depending on crystallisation conditions such as pH, temperature, concentration and nature of precipitant, and the presence or absence of additives. In addition to the macroscopic variety and the quality of diffraction of the crystals, different crystal forms show important differences in the packing of protein molecules. It was noted by Axelsen et al. [1] that, due to crystal contacts, the entrance to the active-site gorge of every monomer in the trigonal crystal form of Torpedo californica acetyl-cholinesterase (TcAChE) is tightly blocked by a symmetry-related molecule.

For Christmas 1939 a girl friend presented me with a book token — I used it to buy Linus Pauling’s newly published Nature of the Chemical Bond 1 which I would otherwise not have been able to afford. I found in it the following passage: “Although the hydrogen bond is not strong it has great significance in determining the properties of substances. Because of its small bond energy and the small activation energy involved in its formation and rupture, the hydrogen bond is especially suited to play a part in reactions occurring at normal temperatures. It has been recognized that hydrogen bonds restrain protein molecules to their native configurations, and I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.”

Molecular electronic devices (MED) have attracted many researchers in both academia and industries since Dr. Forrest Carter of Naval Research Laboratory (USA) organized 1st International Workshop on MED in 1981 (Carter, 1982). A number of international conferences, symposia, forums and/or workshops have been organized since then (Table 1), and various approaches to realize MED have been discussed from the viewpoint of molecular design, molecular assembly, characterization and device design. However, still there are a lot of problems to show a prototype device. When biological molecules including proteins and enzymes are used instead of synthetic organic molecules, MED can be called bioelectronic devices (BED). Compared to MED, BED have shown some outputs such as biosensors, but this is far from the real molecular electronics.

Life in the universe as we know it is based upon systems of cells: from single-cell bacteria to trillions of complexly interacting cells -an organism. It is sometimes instructive to consider these cellular units as being “factories” containing various molecular machines consuming energy and material while carrying out complex processes and manufacturing new materials (Alberts et al., 1994). Interestingly, many of the components of cells -amino acids to proteins to protein complexes (supramolecules) to cellular organelle -are formed by self-assembly under the instructions of DNA. These cells also produce all of the proteins and supramolecules necessary to form tissue and ultimately organisms that are a million-times larger than the original simple protein.

Studies of the structures and amino-acid sequences of proteins, and the relationships between structure and function, are increasing rapidly in terms of the quantity of information available, the number of groups engaged in the field and the areas in which the resultant knowledge is being applied. An obvious impetus results from the projects to determine the entire genetic constitution of humans and other species, which demands interpretation in terms of the proteins which the chromosomal nucleic acid encodes. Many such proteins have been characterised solely as the translations of open reading frames of the nucleic acid code and are likely to be of unknown function and 3-dimensional structure. It seems clear, however, that protein molecules and their constituent domains belong to families that have evolved from a common ancestor and that there may well only be between one and two thousand such families. This number should be compared to the number of proteins for which sequences are so far known (over 80000 in the current release of the OWL composite, non-redundant database (Akrigg et a1..1992; Bleasby & Wootton, 1990) and the 500 or so different proteins whose 3-dimensional structures are known.

The problem of searching for functional sites in protein molecules is far from solving. The straight experimental way is to isolate molecules of both receptor and ligand, co-crystallize them, obtain X-ray data of this complex, analyze it and directly find interacting sites [Burt et al. 1991]. Unfortunately in most cases this ideal could not be reached because of known difficulties in cloning, obtaining, purification, crystallization etc. of both components. This method provides full information about structures of recognition sites but not about particular biological functions of amino acids constituting these sites (e.g. binding, or signal transduction etc.). The usual way to obtain functional information is changing the structure of selected protein sites (via site-directed mutagenesis or chemical modification) followed by comparative biological testing. Step-by-step mutagenesis of the whole protein molecule remains difficult and time-consuming task. Selection of proper mutagenesis strategy makes it enormously multi-variant, although some simplistic approaches (e.g. alanine mutagenesis) could be successful. Thus it is preferential to find probable functional sites and start mutagenesis from it.

The term ‘fish proteases’ refers to hydrolytic enzymes from aquatic species that catalyze degradation of peptide bonds in protein molecules. Proteases play a vital role in biotechnology, food processing and other industries, as well as in a variety of physiological processes (Table 6.1). There is considerable demand for enzymes with the right combination of properties for specific applications in industry. Worldwide sales of industrial enzymes were estimated at about $1.5 billion for 1990 with approximately 48% of all these sales deriving from proteases (Knorr and Sinskey, 1985). Proteases used in industry are mainly derived from plant, animal and microbial sources, whereas their counterparts derived from marine and other aquatic sources have not been extensively used. Some of the reasons for the under-utilization of fish proteases include the following: 1. relatively few studies have been carried out on these enzymes, hence there is limited information about their potential; 2. the seasonal nature of the source material which precludes supply in a steady manner; 3. the unfavorable attitude by workers to the ‘odors’ associated with the source material (i.e. fish offal).

Many studies in recent years have been directed towards producing fully synthetic peptide immunogens (Francis and Clarke 1989; Francis 1993, this volume) which contain the necessary B- and T-cell epitopes for immunogenicity in vivo. However, there is little doubt that carrier proteins still offer the most straightforward and convenient way of presenting poorly immunogenic peptides to the immune system. This may be achieved either by chemical linkage of synthetic peptides to “foreign” protein molecules or by direct linkage to proteins for expression as recombinant fusion proteins. However it should be recognised that the method used to link the peptide to the carrier can greatly influence both the quantitative and qualitative nature of the immune response observed.

Water is one of the most important molecules necessary to sustain life. It is the only substance found in all three physical states. The interaction of water with other substances is critical to most chemical and biological reactions. Proteins also play an important role in many life processes. They provide structure to living cells and regulate many cellular functions. The interaction of these two important molecules plays an important role in determining the functional properties of many food ingredients. The focus of this chapter is to discuss the types of bonding which occur between water and protein molecules and to relate the interaction between water and protein molecules to the functional properties of proteins in foods.

Viruses are highly organized submolecular systems. Their basic components are nucleic acid and protein. More than half of the infectious diseases known at present are caused by them. The nucleic acid, which carries the whole complement of information necessary for generation of the virion, is enclosed for the majority of spherical viruses (Fig. 1) in an icosahedral capsid composed of 60T quasiequivalent protein molecules. Here T is the number of triangulations. Besides the protective function, the capsid is responsible for the sphericity of the virion. Thus, x-ray structural analysis of the biocrystals is a fundamental technique and one of the most informative methods for study of the structure—activity relation of viruses.

The assignment of resonances in the complex spectrum of a protein is still one of the bottlenecks in the application of NMR spectroscopy to the study of the structure and dynamics of protein molecules in solution. During the last few years there has been great interest in the automation of the sequential assignment procedure. It is clear from several of the papers contained in this volume that progress is being made in the automation of many aspects of the spectral-assignment process. In this paper a specific aspect of automated assignment, dealing with the assignment of the spectra of homologous proteins, will be discussed.

In the 1970-s Davydov proposed a soli ton mechanism of energy transport in biological macromolecules1. At present the concept of the “Davydov soliton” has been used in many studies. In particular, the Davydov soliton has been put into operation as a mechanism useful in the description of a variety of biological phenomena2, 3. Numerical studies by Scott and co-workers2−4 supported the appearance of Davydov solitons in Alpha-helix protein molecules. Therefore an experimental detection of solitons appear to be natural.

Generally speaking, similarities of relaxation in glasses and biomolecules are due to the fact that in both cases a very large number of molecular configurations is involved in the relaxation processes1. A characteristic feature of relaxation in such complex systems is the non-exponential time dependence of the relaxation functions; in the case of glasses the measured relaxation functions are usually well described by Kohlrausch’s fractional-exponential formula. One of the first models for non-exponential relaxation in glasses and undercooled melts was Glarum’s defect-diffusion model2. An extension of the model, in which the molecular units visited by a defect relax with a finite rate, is presented. It is shown that a related defect-diffusion model can be applied successfully to the relaxation processes in ionic channels in protein molecules, which lead to a non-exponential distribution of the closed times of the channels. Considering the special case of a closed-time distribution following a power law, a general model for the gating kinetics of ionic channels is formulated, which is characterized by a waiting-time distribution. The waiting-time distribution is found to follow the same power law.

Total body protein has often been estimated from the total body potassium (TBK) content. The basic assumption is that there exists a constant ratio between potassium and cellular nitrogen. In healthy persons the intracellular nitrogen constitutes about 65 % of total body nitrogen (TBN). In some diseases, such as cancer, the TBK and TBN may vary in different ways. It has been shown (Jeejeebhoy et al., 1982) that TBK and TBN do not maintain the same ratio in malnutrition nor during total parenteral nutrition (TPN). Assuming that almost all of the body nitrogen is bound to protein molecules, the best way to estimate total body protein is by estimation of TBN content (Burkinshaw, 1987). The aim of this study was to investigate changes in TBK and TBN in patients with testicular carcinoma treated with cis-platin, with and without TPN.

The precise developmental mechanisms leading to myoblast fusion and the subsequent functional relationships that exist amongst the myonuclei contained within each syncytium are for the most part ill-defined. In contrast, extraordinary advances have been achieved in defining the precise molecular deficits causing several inherited diseases of muscle. In such diseases, functional rescue of muscle might be achievable by the introduction of genetically normal myonuclei but this strategy ultimately depends upon the interrelationships that exist between the myonuclei and the syncytial cytoplasm. If the mRNA encoded by each myonucleus was translated into protein molecules that were subsequently restricted to a “nuclear territory”, rescue of such mosaic fibers might depend upon not only the number of transplanted myonuclei but also their intrafiber distribution. Alternatively, if proteins encoded by each myonucleus were distributed uniformly throughout the syncytium, the spatial organization of the transplanted myonuclei could be irrelevant.

Various electrophysiological properties of skeletal muscles have been extensively studied in adult vertebrates (Hodgkin and Horowicz, 1959; Nakajima et al., 1962; Adrian et al., 1970; Campbell and Hille, 1976; Campbell, 1976; Beaty and Stefani, 1975; Sanchez and Stefani, 1978). Characteristics of ion channels activated by a transmitter, acetylcholine, have also been the subject of exhaustive studies (Katz and Miledi, 1972; Anderson and Stevens, 1973; Neher and Sakmann, 1976; Takeuchi and Takeuchi, 1960; Maeno et al., 1977). All of these distinct membrane properties may be regulated by and expressed through large protein molecules that reside in the lipid bilayer of the sarcolemma (Singer and Nicholson, 1972). These molecules may be inserted into the membrane de novo or may be activated during membrane differentiation at certain developmental stages.

A number of structural and chemical investigations (1–6) have shown that, even though protein molecules possess a high degree of secondary structure, they have numerous component segments which exhibit substantial fluctuations from their native conformations. A majority of these fluctuations represent a localized denaturation of small portions of the structure and result from the natural equilibrium between protein-protein and protein-solvent interactions. Although most of these fluctuations probably have little direct effect on the regions responsible for functional activity of the protein, a growing body of evidence indicates that conformational change is an important ingredient in a significant number of biological processes (7–10).

Evidence for phylogeny derives basically from morphological comparisons, whether of the structure of protein molecules, chromosomes, skulls, or, in the case of paleontological material, mainly teeth. The concept of homology arises from the recognition that some resemblances are more significant than others that are superficial or accidental. It is necessary to decide, when comparing two species, which structural element in one species should be compared with an element in the other. Such corresponding structures in different animals are given the same name (Gr. homos, same; logos, word), and resemblances and differences between them form the raw data on which phylogenetic hypotheses are based. Whether resemblances are due to inheritance from a common ancestor, or whether they have been produced by parallel evolution, is a question that can be decided only after considerable investigation, if at all. Therefore, in this paper I will use the term homology in its etymological sense, to mean morphologically comparable structures, whether or not their resemblance is due to common ancestry.

Protein molecules and crystals formed of them are unique objects which differ in many respects from ordinary organic molecules and crystals of organic and inorganic substances. A globular protein molecule is a system consisting of 103-104 atoms, built according to the genetic information inherited by a given organism; assembly of the molecules proceeds at the self-organization level. Globular protein molecules mav be crystallized, and the crystals thus obtained have a number of striking features which will be discussed below. One has to mention immediately that the protein molecules may associate not only into three-dimensional, triply periodic crystals but also into two—dimensional and tubular crystals, as well as into pseudospherical shells with point symmetry.

Dietary protein antigens including wheat, cows milk proteins, and ovalbumin have been identified in breast milk of lactating women (1–3). The quantity of antigen reported has varied depending on the individual study. Wheat (3) and cows milk proteins (1) were detected by immunoprecipitation at concentrations of about 1 µg/ml, while ovalbumin (OVA) and beta-lacto-globulin (a specific cow’s milk protein not present in human serum or milk) were detected in the range of 100 pg/ml to 6 ng/ml (2). Methodologic variations have been suggested to account for these differences in dietary antigen levels in breast milk (2). An alternate hypothesis is that inherent differences in the protein molecules themselves, in their processing in the lactating female or in their mechanism(s) of transport into breast milk, may account for the differences in reported concentrations. In the present experiments, we examined the pattern of clearance of four radiolabeled protein antigens from maternal circulation in the mouse, and their localization and catabolism within selected tissues.

The fashion by which glycoproteins or, in general, protein molecules in plasma are eliminated from the circulation and eventually degraded is at present unknown. A vast amount of work on protein turnover, mainly pertaining to albumin, has established that unlike more complicated organized structures (i.e., cells) proteins have no definite life span and each protein molecule, regardless of age, has statistically the same chance of being eliminated.1

Evolutionists have turned to molecular studies because nucleic acids are genetic substances that carry evolving organisms from one cell generation to the next, and protein molecules are the mirrored reflection of some of the structure of these nucleic acids.

Current trends in molecular biology argue convincingly in favor of a precise relation between biologic function and shape of macromolecules. This is especially relevant in the case of heparin; the evidence points to a specific inhibition by this highly charged polysaccharide of protein molecules involved in blood coagulation. Thus, information concerning the molecular structure and shape of the heparin molecule should be considered as an essential step forward in understanding the biologic mechanisms involved in coagulation.

Intracellular proteolysis is necessary to adapt the protein composition of cells to environmental changes, to correct errors in transcription or in traslation, to degrade inactive pre- and pro-peptides and to liberate amino acids as an energy source from superfluous protein molecules in case of need1.

The introduction of Nuclear Magnetic Resonance as a second method for protein structure determination, besides X-ray diffraction in single crystals, has already helped to significantly increase the number of known protein structures. However, it is also of more fundamental interest, since it provides data that are in many ways complementary to those obtained from X-ray crystallography. Its use thus promises to widen our view of protein molecules with regard to a better grasp of the relations between structure and function. The complementarity of the two methods results from the facts that the time scales of the two types of measurements are widely different, and that, in contrast to the need of single crystals for diffraction studies, the NMR measurements use proteins in solution or other noncrystalline states.

Organisms in the three major classes of annelids, the Polychaetes (marine sandworms), the Oligochaetes (earthworms), and the Hirudinea (leeches), contain at least three types of collagenous structures. These structures are (1) handed collagen fibers that are morphologically identical to those observed in vertebrate and many invertebrate species, (2) basal laminae which are common to all metazoan organisms, and (3) non-banded cuticle fibers which are found in the annelids and nematodes. The cuticle collagens are the longest protein molecules known, with documented lengths of up to 2,400 nm for the native molecules which form large unhanded fibers and are not crosslinked. This chapter will review the morphological and biochemical studies of the collagens of these three classes of annelids and will relate these collagens to vertebrate collagen types where appropriate.

One of the most fascinating biochemical processes is the biosynthesis of protein molecules. This intricate and accurate process takes place in all organisms on their ribosomes in a similar manner. It involves specific interactions of the ribosomes with mRNAs, aminoacyl-tRNAs and a number of proteins such as initiation, elongation and termination factors. During this process the ribosomes dissociate into their two subunits which reassociate after the initiation process. Each of these subunits is a structurally independent distinct assembly of many proteins and several RNA chains.

Currently there is a renewed interest in the use of hyperthermia as a treatment modality in the management of malignant disease. This interest is based on old, anecdotal data, as well as on recent quantitative investigations (reviews: Dietzel, 1975; Thrall et al., 1976; Har-Kedar and Bleehen, 1976; Bronk, 1976; Gerner et al., 1975). The mode of cell killing by hyperthermia is not well understood. Thermodynamic considerations suggest that protein molecules are at risk, and some authors have focused on chromosomal proteins (review: Dewey, 1977). Other evidence implicates RNA synthesis (Mondovi et al., 1969) or membranes (Bowler et al., 1973; Hahn et al., 1975). Survival curves of mammalian cells exposed to elevated (> 42°C) temperatures for variable lengths of time resemble curves for cells exposed to ionizing radiations, i.e., a shoulder at short exposure times followed then by an exponential part at longer exposure times.

The biological importance of charge-transfer reactions involving structural proteins is discussed. Particular emphasis is placed on those reactions which result in the generation of delocalized electronic charges in the protein molecules, and several experimental studies on various protein-methylglyoxal complexes are described which provide strong evidence for the existence of such a process. Such studies have led to the synthesis of a new compound that exhibits interesting physiological activity.

Collagen is the family name of a group of genetically distinct protein molecules that has evolved as the major fibrous component of the extra-cellular matrix. The other components of this matrix — the weight fractions of which depend upon the function of the particular tissue — include the proteins elastin and keratin, mucopolysaccharides, proteoglycans, and lipids irrigated by a solution containing a number of inorganic ions notably Na+, Cl-, Ca++, K+, vitamins and free oxygen.

The concept of crosslinkages as the base for aging was suggested by Bjorksten (1941), who in 1942 expressed this theory as follows: “The aging of living organisms I believe is due to the occasional formation by tanning, of bridges between protein molecules, which cannot be broken by the cell enzymes. Such irreparable tanning may be caused by tanning agents foreign to the organism or formed by unusual biological side reactions, or it may be due to the formation of a tanning bridge in some particular position in the protein molecule. In either event, the result is that cumulative tanning of body proteins which we know as old age.” If we add the words “and nucleic acids” to “proteins” this still covers the tenets of the crosslinking theory.

Gregorio Weber pointed out over 25 years ago (Weber, 1961) that there are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and function of protein molecules. He and his co-workers have led the way in finding new ways to use intrinsic and extrinsic fluorescent probes, with both steady-state and nanosecond time-resolved techniques, to investigate the static and dynamic structures of biological macromolecules. We are thankful to Gregorio, not only for pointing us in the right scientific direction, but also for showing us, by example, how scientists ought to interact with each other. This continues to make biochemical fluorescence an enjoyable field to work in.

It is generally recognised that protein molecules are arrangend into small folding units domains. We now have a sufficiently large sample of three dimensional structures to realise that there is only a limited number of different folding patterns for such domains (Richardson, 1981). The most common type of these patterns are the α/s domains which comprise around 25% of all known domain structures. The central fold of these domains is \/ery simple consisting of a number of hydrogen bonded parallel strands joined by helices. The strands all have their NH2 ends at the same edge of the resulting s-sheet. The sheet thus has a polarity with an NH2-edge and a C00H edge. Loop regions, which are usually relatively short, join the strands to the helices at both ends.

Our knowledge of the influences of diseased states on protein metabolism in brain and the repercussion on the protein molecules remains meager. The problem is very difficult: each cell contains at least 800 different kinds of proteins and a change in one or the addition of one more may not be measurable with our present methods.