Professor Tatsuo Miyazawa: from molecular structure to biological function

The late Prof. Tatsuo Miyazawa was an outstanding physical chemist, who established a number of spectroscopic methods to analyse the structures of proteins, peptides and nucleotides, and used them to understand molecular functions. He developed an infrared spectroscopic method to quantitatively analyse the secondary structures, α-helices and β-strands, of proteins. He successfully utilized nuclear magnetic resonance (NMR) methods to determine the conformations of peptides and proteins, particularly with respect to the interactions with their target molecules, which served as a solid basis for the wide range of applications of NMR spectroscopy to life science research. For example, he found that physiologically active peptides are randomly flexible in solution, but assume a particular effective conformation upon binding to their functional environments, such as membranes. He also used NMR spectroscopy to quantitatively analyse the conformer equilibrium of nucleotides, and related the dynamic properties of the modified nucleosides naturally-occurring in transfer ribonucleic acids (tRNAs) to their roles in correct codon recognition in protein synthesis. Furthermore, he studied the mechanisms of protein biosynthesis systems, including tRNA and aminoacyl-tRNA synthetases. Inspired by the structural mechanism of amino acid recognition by aminoacyl-tRNA synthetases, as revealed by NMR spectroscopy, he initiated a new research area in which non-natural amino acids are site-specifically incorporated into proteins to achieve novel protein functions (alloprotein technology).     

Recognition of Membrane-Bound Fusion-Peptide/MPER Complexes by the HIV-1 Neutralizing 2F5 Antibody: Implications for Anti-2F5 Immunogenicity

The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen.     

Electron crystallography of aquaporins

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.     

Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX)

Oxygenic photosynthesis depends on a highly conserved electron transport system, which must be particularly dynamic in its response to environmental and physiological changes, in order to avoid an excess of excitation energy and subsequent oxidative damage. Apart from cyclic electron flow around PSII and around PSI, several alternative electron transport pathways exist including a plastoquinol terminal oxidase (PTOX) that mediates electron flow from plastoquinol to O(2). The existence of PTOX was first hypothesized in 1982 and this was verified years later based on the discovery of a non-heme, di-iron carboxylate protein localized to thylakoid membranes that displayed sequence similarity to the mitochondrial alternative oxidase. The absence of this protein renders higher plants susceptible to excitation pressure dependant variegation combined with impaired carotenoid synthesis. Chloroplasts, as well as other plastids (i.e. etioplasts, amyloplasts and chromoplasts), fail to assemble organized internal membrane structures correctly, when exposed to high excitation pressure early in development. While the role of PTOX in plastid development is established, its physiological role under stress conditions remains equivocal and we postulate that it serves as an alternative electron sink under conditions where the acceptor side of PSI is limited. The aim of this review is to provide an overview of the past achievements in this field and to offer directions for future investigative efforts. Plastoquinol terminal oxidase (PTOX) is involved in an alternative electron transport pathway that mediates electron flow from plastoquinol to O(2). This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

The need for a shared database infrastructure: combining X-ray crystallography and electron microscopy

Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases. Received: 26 January 2000 / Revised version: 15 May 2000 / Accepted: 15 May 2000     

Vesicles and particles of sodium bis(2-ethylhexyl) phosphate in binary and ternary systems

Vesicles were identified in aqueous solution of pure sodium bis(2-ethylhexyl) phosphate, a short branched chain surfactant. Superficial tension measurements show that the vesicles appear above a molality of 0.02 (0.69 %w). These aggregates are equilibrium structures. The "packing parameter' theory established by Israelachvili et al. allows the prediction of the occurrence of such vesicles. If an organic solvent, such as xylene or ethylhexanoate, is added to the binary system, a different type of aggregate appears, the size of which is determined by several methods including electron microscopy and light scattering. Interfacial tension measurements show that these aggregates would be expected to form above a molality of 0.02. According to our experimental results, the microstructure of these aggregates can be described as micelles and/or vesicles, swollen or not.     

John Newport Langley and His Construction of the Autonomic (Vegetative) Nervous System

The paper describes the scientific career of Professor John Newport Langley, an outstanding English physiologist and histologist, member of the London Royal Society and its Vice-President. The main scientific achievements of Langley are dealing with anatomy and physiology of the autonomic (vegetative) nervous system that he considered as an entirely efferent system. The autonomic nervous system was divided by Langley into three parts: sympathetic, parasympathetic, and enteral (intestinal) systems. He has also established functional differences of the autonomic nervous system from the somatic one. He suggested the presence of synaptic contacts on muscle and secretory cells, as well as the existence in these synaptic structures of a peculiar receptor substance providing interaction of nerve fibers with postganglionic neurons and effector cells of visceral organs. The examples of development of several Langley's concepts at present are given.     

Govindjee at 80: more than 50 years of free energy for photosynthesis

We provide here a glimpse of Govindjee and his pioneering contributions on the two light reactions and the two pigment systems, particularly on the water–plastoquinone oxido-reductase, Photosystem II. His focus has been on excitation energy transfer; primary photochemistry, and the role of bicarbonate in electron and proton transfer. His major tools have been kinetics and spectroscopy (absorption and fluorescence), and he has provided an understanding of both thermoluminescence and delayed light emission in plants and algae. He pioneered the use of lifetime of fluorescence measurements to study the phenomenon of photoprotection in plants and algae. He, however, is both a generalist and a specialist all at the same time. He communicates very effectively his passion for photosynthesis to the novice as well as professionals. He has been a prolific author, outstanding lecturer and an editor par excellence. He is the founder not only of the Historical Corner of Photosynthesis Research, but of the highly valued Series Advances in Photosynthesis and Respiration Including Bioenergy and Related Processes. He reaches out to young people by distributing Z-scheme posters, presenting Awards of books, and through tri-annual articles on “Photosynthesis Web Resources”. At home, at the University of Illinois at Urbana-Champaign, he has established student Awards for Excellence in Biological Sciences. On behalf of all his former graduate students and associates, I wish him a Happy 80th birthday. I have included here several tributes to Govindjee by his well-wishers. These write-ups express the high regard the photosynthesis community holds for “Gov” and illuminate the different facets of his life and associations.     

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.     

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.     

Field emission scanning electron microscopy of plant cells

Summary Two basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution three-dimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.Abbreviations DMSO dimethylsulfoxide - FESEM field emission scanning electron microscope (-scopy) - MTSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - SEM scanning electron microscope (-scopy) - TEM transmission electron microscope (-scopy)     

Aldolase-localization in cultured cells: cell-type and substrate-specific regulation of cytoskeletal associations.

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20-2000 microM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20-60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin-aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.     

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA’s are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.     

Vincenzo Malacarne (1744-1816): a researcher in neurophysiology between anatomophysiology and electrical physiology of the human brain

Since his first years at Turin until the last years of his life at Padua, Vincenzo Malacarne devoted most of his time to the examination of the structures and the various parts of which the cerebellum and the human brain are composed. He is rightly considered as one of the first to have correctly described the anatomy of the cerebellum, as well in the field of human anatomy and comparative anatomy. However, his work cannot be reduced to these studies. He worked out a cerebral physiology, with organic and intellectual phenomena in mind, established on an anatomopsychic parallelism. This parallelism is itself founded on a rational and mathematical criterion: the number of lamellae contained in the cerebellum. A letter written by him in 1792 and addressed to Abbot Denina was recently found by the present author in November 2005 at the Academy of Sciences of Turin. Malacarne exposed his project of studying the animal electricity put forward by Galvani within the cerebral organ. May it be that Malacarne had in mind the physiology of his time while trying to record an electric activity within the brain?     

Ultrastructure analysis of cadmium-tolerant and -sensitive cell lines of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Previously, a stable cell suspension culture of cucumber tolerant to cadmium (Cd) was established (Gzyl and Gwóźdź, Plant Cell Tissue Organ Cult 80:59–67, 2005). In this study, ultrastructures of Cd-tolerant and -sensitive cells were analyzed by transmission electron microscopy (TEM). Ultrastructural differences between cell lines exposed to 100 μM CdCl₂ were observed both at cellular and organelle levels. Tolerant cells exposed to Cd exhibited well-preserved cellular structures in comparison with sensitive cells. Increased numbers of osmiophilic globules in the cytoplasm and nucleolus-associated bodies as well as electron dense material in vacuoles were observed in cadmium tolerant cells. In contrast, ultrastructure of sensitive cells following exposure to Cd exhibited distinct disturbances including vacuolation, disintegration of cytoplasm, and structural changes in both mitochondria and endoplasmic reticulum. TEM observations confirmed the adaptation of tolerant cells to Cd.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Single particle reconstruction of membrane proteins: A tool for understanding the 3D structure of disease-related macromolecules

Membrane proteins play important roles in cell functions such as neurotransmission, muscle contraction, and hormone secretion, but their structures are mostly undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Electron microscopy-based single particle reconstruction, a computer-aided structure determination method, reconstructs a three-dimensional (3D) structure from projections of monodispersed protein. A large number of particle images are picked up from EM films, aligned and classified to generate two-dimensional (2D) averages, and, using the Euler angle of each 2D average, reconstructed into a 3D structure. This method is challenging due to the necessity for close collaboration between classical biochemistry and innovative information technology, including parallel computing. However, recent progress in electron microscopy, mathematical algorithms, and computational ability has greatly increased the subjects that are considered to be primarily addressable using single particle reconstruction. Membrane proteins are one of these targets to which the single particle reconstruction is successfully applied for understanding of their structures. In this paper, we will introduce recently reconstructed channel-related proteins and discuss the applicability of this technique in understanding molecular structures and their roles in pathology.     

Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.