1. Introduction 310

2. Protein-only hypothesis 312

3. The scrapie prion protein PrPSc313

3.1 Purification of PrP 27–30 313

3.2 Proteinase K resistance 314

3.3 Scrapie-associated fibrils 314

3.4 Smallest infectious unit 316

3.5 Conformational properties 316

3.6 Dissociation and stability 319

4. The cellular prion protein PrPC321

4.1 Prnp expression 321

4.2 Biosynthetic pathway 322

4.3 NMR structures 324

4.4 Copper binding 326

5. Post-translational PrP conversion 327

5.1 Conformational isoforms 327

5.2 Location of propagation 329

5.3 Minimal PrP sequence 330

5.4 Prion species barrier 331

5.5 Prion strains 332

6. Effect of familial TSE mutations 333

6.1 Thermodynamic stability of PrPC 334

6.2 De novo synthesis of PrPSc 335

6.3 Transmembrane PrP forms 337

7. Physical properties of synthetic PrP 337

7.1 Amyloidogenic peptides 337

7.2 Folding intermediates 339

8. Hypothetical protein X 340

8.1 Two species-specific epitopes 340

8.2 Mapping the protein X epitope 341

9. Chaperone-mediated PrP conversion 343

9.1 Hsp60 and Hsp10 chaperonins 343

9.2 GroEL promoted PrP-res formation 345

9.3 Membrane-associated chaperonins 345

9.4 Preference of GroEL for positive charges 347

9.5 Potential GroEL/Hsp60 epitopes on PrP 347

9.6 Conformations of chaperonin-bound PrP 349

9.7 Conserved Hsp60 substrate binding sites 349

9.8 Requirement of ATP-hydrolysis 351

9.9 Hsp60-mediated prion propagation 354

10. Template-assisted annealing model 355

11. Acknowledgments 357

12. References 357

Although the central paradigm of protein folding (Anfinsen, 1973), that the unique three-dimensional structure of a protein is encoded in its amino acid sequence, is well established, its generality has been questioned due to two recent developments in molecular biology, the ‘prion’ and ‘molecular chaperone’. Biochemical characterization of infectious scrapie material causing central nervous system (CNS) degeneration indicates that the necessary component for disease propagation is proteinaceous (Prusiner, 1982), as first outlined by Griffith (1967) in general terms, and involves a conversion from a cellular prion protein, denoted PrPC, into a toxic scrapie form, PrPSc, which is facilitated by PrPSc acting as a template for PrPC to form new PrPSc molecules (Prusiner, 1987). The ‘protein-only’ hypothesis implies that the same polypeptide sequence, in the absence of any post-translational modifications, can adopt two considerably different stable protein conformations (Fig. 1). Thus, in the case of prions it is possible, although not proven, that they violate the central paradigm of protein folding. There is some indirect evidence that another factor, provisionally named ‘protein X’, might be involved in the conformational conversion process (Prusiner et al. 1998), which includes a dramatic change from α-helical into β-sheet secondary structure (Fig. 1). This factor has not been identified yet, but it has been proposed that protein X may act as a molecular chaperone. The idea that molecular chaperones play a critical role in the generation of PrPSc is appealing also from a theoretical point of view, because PrPSc formation involves changes in protein folding and possibly intermolecular aggregation (Fig. 1), processes in which chaperones are known to participate (Musgrove & Ellis, 1986). The discovery and functional analysis of more than a dozen molecular chaperones made it clear that these proteins do not complement folding information that is not already contained in the genetic code (Ellis et al. 1989); rather they facilitate the folding and assembly of proteins by preventing misfolding and refolding misfolded proteins (Hartl, 1996). Whether a molecular chaperone or another type of macromolecule is identified as the conversion factor, therefore, the molecular chaperone concept is likely to contribute to the understanding of the molecular nature of PrPC to PrPSc conversion.

In this review I consider the prion concept from the view of a structural biologist whose main interest focuses on spontaneous and chaperone-mediated conformational changes in proteins.

Customized silicon diode detectors are widely used for elementary particle detection for their good spatial resolution. Silicon detectors are also excellent X-ray detectors in the energy range of interest for applications in synchrotron radiation experiments and, with small elements laid out in a two-dimensional array, may provide high performance imaging devices for future diffraction experiments.

Night-migratory songbirds appear to sense the direction of the Earth's magnetic field via radical pair intermediates formed photochemically in cryptochrome flavoproteins contained in photoreceptor cells in their retinas. It is an open question whether this light-dependent mechanism could be sufficiently sensitive given the low-light levels experienced by nocturnal migrants. The scarcity of available photons results in significant uncertainty in the signal generated by the magnetoreceptors distributed around the retina. Here we use results from Information Theory to obtain a lower bound estimate of the precision with which a bird could orient itself using only geomagnetic cues. Our approach bypasses the current lack of knowledge about magnetic signal transduction and processing in vivo by computing the best-case compass precision under conditions where photons are in short supply. We use this method to assess the performance of three plausible cryptochrome-derived flavin-containing radical pairs as potential magnetoreceptors.

To date, most antibodies from combinatorial libraries have been selected purely on the basis of binding. However, new methods now allow selection on the basis of function in animal cells. These selected agonist antibodies have given new insights into the important problem of signal transduction. Remarkably, when some antibodies bind to a given receptor they induce a cell fate that is different than that induced by the natural agonist to the same receptor. The fact that receptors can be functionally pleiotropic may yield new insights into the important problem of signal transduction.

The code of inheritable information which directs the synthesis of protein molecules is now known. This information is contained in the replicative molecules deoxyribonucleic or ribonucleic acid (Avery, Macleod & McCarty, 1944; Loeb & Zinder, 1961). Translation of the nucleic acid ‘information’ into protein molecules involves the initial transcription of the nucleic acid molecules into transient messenger ribonucleic molecules which are single stranded base paired complements of the more stable ‘information’ nucleic acid molecules. The events leading to the translation of the linear order of nucleic acid information in mRNA to a corresponding linear sequence of amino acids in a protein are largely elucidated at this time and will be the subject of this discussion. Since the knowledge of the genetic code is a result of data obtained from in vitro translational studies which employ the equivalent of mRNA, the genetic code is described as the RNA code rather than its DNA complement. Elucidation of the RNA code has been the focus of research activity for a large number of research laboratories over the past 10–15 years. As a result of these investigations we have a detailed description of the genetic code for life on this planet. This code appears universal in its organization and translational mechanism. Since the interactions of macromolecules necessary for genetic translation are understood with greater precision than the factors responsible for the origin of the Genetic Code, this discussion will deal in more detail with the events in the translation of genetic information.

The non-invasive and non-destructive aspects of NMR and ESR spectroscopy have prompted a variety of research on the pathophysiological impact of murine malaria. NMR is unique in its ability to monitor intracellular pH non-invasively in a heterogeneous sample, a compartmentalized cell and in a whole organism. It has also been shown to be sensitive to unusual structures and metabolic products in free-living protozoa such as Acanthamoeba (Deslauriers et al. 1982a) and Tetrahymena (Deslauriers et al. 1982b; Jarrell et al. 1981). Using the appropriate spin probe, ESR can give valuable information on membrane structure (Schreier, Polnaszek & Smith; 1978). It is particularly useful when quantities of material are limited.

After the work of Marmarelis & Naka (1972, 1973) in the catfish retina, systems analysis using stochastic stimuli has had a boom in the seventies (e.g. McCann & Marmarelis, 1975; Eckert & Bishop, 1975; French & Wong, 1977; Lipson, 1975; McCann, 1974; Naka, Marmarelis & Chan, 1975; Spekreijse & Reits, 1982; Trimble & Phillips, 1978; Terzuolo et al. 1982). White-noise analysis was considered to be a general tool for investigating nonlinear systems gaining a maximum of information with a minimum of assumptions about the system. The modification of the original Wiener theory (Wiener, 1958; Cameron & Martin, 1947; McKean, 1972) by Lee & Schetzen (1965) made the theory fairly easy to implement into widely available computers and thus accessible to a larger number of experimenters.

Most biological processes involve recognition and binding between molecules. Protein-peptide interactions are particularly common as, for example, in the action of hormones, such as oxytocin and vasopressin, or neurotransmitters including morphine-like peptides.

Copper (Cu) is an essential transition metal providing activity to key enzymes in the human body. To regulate the levels and avoid toxicity, cells have developed elaborate systems for loading these enzymes with Cu. Most Cu-dependent enzymes obtain the metal from the membrane-bound Cu pumps ATP7A/B in the Golgi network. ATP7A/B receives Cu from the cytoplasmic Cu chaperone Atox1 that acts as the cytoplasmic shuttle between the cell membrane Cu importer, Ctr1 and ATP7A/B. Biological, genetic and structural efforts have provided a tremendous amount of information for how the proteins in this pathway work. Nonetheless, basic mechanistic-biophysical questions (such as how and where ATP7A/B receives Cu, how ATP7A/B conformational changes and domain–domain interactions facilitate Cu movement through the membrane, and, finally, how target polypeptides are loaded with Cu in the Golgi) remain elusive. In this perspective, unresolved inquiries regarding ATP7A/B mechanism will be highlighted. The answers are important from a fundamental view, since mechanistic aspects may be common to other metal transport systems, and for medical purposes, since many diseases appear related to Cu transport dysregulation.

It is only some 50 years ago that biophysicists obtained reliable experimental methods for estimating the molecular weights of biological macromolecules, chiefly as a result of Svedberg's work in developing the ultracentrifuge as an analytical instrument (Svedberg & Pedersen, 1940). Having gained some understanding of the size of proteins, interest soon thereafter turned to the shape and rotational relaxation times of these molecules, and Perrin's work on fluorescence depolarization helped to lay the foundations there (Perrin, 1929). Biophysics had to wait for the development of X-ray spectroscopy of proteins and nucleic acids to provide a picture of the interior structure of biological macromolecules.

1. Viruses and other informational parasites are a ‘fact of life’ 304

2. Evolution drives diversity in viral strategies and host interactions 304

3. Cellular defense often involves recognizing nucleic acid structures that are indicative of unwanted information duplication 305

3.1 Double-stranded RNA (dsRNA) 305

3.2 Single-stranded DNA (ssDNA) 305

3.3 Unspliced mRNAs 306

3.4 Polycistronic mRNAs 306

3.5 Multi-copy DNAs 306

3.6 Other structures 306

4. dsRNA as a case study: building a multilevel response to foreign structure 306

5. Getting there is half the fun: disseminated immunity in RNAi 308

6. Viruses strike back … so why do we still get sick? 308

Cells face a constant struggle against unwanted instructions that arrive in the form of viruses and transposons. At the core of this battle are two issues: how can cellular machinery recognize certain informational molecules as ‘unwanted’ and how can the cell use this recognition to effectively silence malicious genetic activity. While defenses against some specific parasites may be triggered by individual nucleic acid or protein sequences, such sequence-specific mechanisms have the limitation of allowing the parasite to evade following relatively minor evolutionary change. A more general set of defense mechanisms is based on recognition of structural features that are intrinsic aspects of one or more parasitic lifestyle. Recognition of extended regions of double-stranded RNA (dsRNA) provides cells with one such defensive modality. Essentially absent during ‘normal’ gene expression, long stretches of dsRNA within a cell serve as a dramatic warning that a segment of information may be replicating as RNA. In addition to exemplifying many of the mechanistic issues in genome defense, the cellular response to dsRNA provides several examples of the logic by which organisms attempt to focus their limited immunity resources on the most immediate and dangerous targets.

At the end of the last century it was established that the different nerve cells along a neuronal path do not come into direct physical contact with one another, but that there are narrow gaps between them, called synapses (Sherrington, 1897; Ramón y Cajal, 1906). Elliot (1905) made the basic experimental observation that the propagation of nerve impulses across a synapse might be mediated by specific chemical agents (see Fig. i). Such substances are now called neurotransmitters, and some 20 different compounds putatively responsible for synaptic transmission in different parts of the nervous system are known at present, e.g. a few recently isolated polypeptides. The most extensively studied transmitters are acetylcholine and the catecholamine group, consisting of dopamine (a), noradrenaline (b), and adrenaline (c).

1. Introduction 321

2. Tyrosine kinase receptors 322

3. Histidine kinase sensors 325

4. G-protein coupled receptors 327

5. Principles 328

6. Acknowledgments 329

7. References 330

Biological cells need to be responsive to various stimuli, primarily chemical ligands from their environments. Specific receptor molecules embedded in the plasma membrane detect the different biochemical signals that impact the cell, and these receptors are the conduits for transmission of this information to the cell interior for action. There are several classes of signal transduction receptors and many specific receptors within each of the major classes. This review emphasizes the structural biology of three major classes of transmembrane receptors – tyrosine kinase receptors, histidine kinase sensors, and G-protein coupled receptors. Biophysical principles that govern the processes of signal transduction across cell membranes are also discussed.

Many basic events which concern management and manipulation of genes within a cell – for instance transcription, replication and recombination – rely on specific interactions between proteins and nucleic acids. Such interactions are also essential for many house-keeping functions, like packing and unpacking of DNA in chromatin and assembly of ribosomes. Moreover, the details of protein–nucleic acid interplay is essential for understanding the action of viruses. The list of functional mechanisms in biology that rely on protein–DNA and protein–RNA interactions can be made much longer, but these examples represent some of the topics which motivated structural biologists to study complexes between proteins and nucleic acids as a first step beyond structure determinations of individual biomolecules.

1. INTRODUCTION 195

2. THE PROTEIN SYNTHESIS SYSTEM 199

2.1 Aminoacyl-tRNA synthetases 199

2.1.1 Glutaminyl-tRNA synthetase 202

2.1.2 Glutamyl-tRNA synthetase 206

2.1.3 Tyrosyl- and tryptophanyl-tRNA synthetases 207

2.1.4 Methionyl-tRNA synthetase 208

2.1.5 Aspartyl-tRNA synthetase 208

2.1.6 Lysyl-tRNA synthetase 209

2.1.7 Seryl-tRNA synthetase 210

2.1.8 Glycyl- and histidyl-tRNA synthetases 210

2.1.9 Phenylalanyl-tRNA synthetase 211

2.2 Ribosomal proteins 211

2.2.1 L7/L12 212

2.2.2 L30 214

2.2.3 S5 215

2.2.4 S17 215

2.2.5 L6 215

2.2.6 L9 216

2.2.7 S6 217

2.2.8 L1 217

2.2.9 L14 217

2.2.10 S8 217

2.3 Elongation factors 218

2.3.1 EF-Tu 218

2.3.2 EF-G 219

3. SPLICEOSOMAL PROTEINS 221

3.1 U1 snRNP protein A 222

4. PROTEINS FROM RNA VIRUSES 223

4.1 Viral enzymes and regulatory proteins 224

4.1.1 Reverse transcriptase 224

4.1.2 Tat 224

4.1.3 Rev 225

4.2 Viral capsid proteins 225

4.2.1 Tobacco Mosaic Virus 226

4.2.2 Satellite Tobacco Mosaic Virus 226

4.2.3 Bean-Pod Mottled Virus 226

4.2.4 Black Beetle Virus and Flock House Virus 228

4.2.5 Bacteriophage MS2 228

5. OTHER RNA-BINDING PROTEINS 230

5.1 tRNA-guanine transglycosylase 230

5.2 Major cold-shock protein 230

5.3 Rop 231

5.4 Ricin 231

6. CONCLUSION 232

7. ACKNOWLEDGEMENTS 233

The aggregation of the 11 residue long NACore peptide segment of α-synuclein (68-GAVVTGVTAVA-78) has been investigated using a combination of cryogenic transmission electron microscopy (cryo-TEM), small- and wide-angle X-ray scattering, and spectroscopy techniques. The aqueous peptide solubility is pH dependent, and aggregation was triggered by a pH quench from pH 11.3 to approximately pH 8 or 6, where the average peptide net charge is weakly negative (pH 8), or essentially zero (pH 6). Cryo-TEM shows the presence of long and stiff fibrillar aggregates at both pH, that are built up from β-sheets, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. The fibrils are crystalline, with a wide angle X-ray diffraction pattern that is consistent with a previously determined crystal structure of NACore. Of particular note is the cryo-TEM observation of small globular shaped aggregates, of the order of a few nanometers in size, adsorbed onto the surface of already formed fibrils at pH 6. The fibrillation kinetics is slow, and occurs on the time scale of days. Similarly slow kinetics is observed at both pH, but slightly slower at pH 6, even though the peptide solubility is here expected to be lower. The observation of the small globular shaped aggregates, together with the associated kinetics, could be highly relevant in relation to mechanisms of secondary nucleation and oligomer formation in amyloid systems.

In neurodegenerative diseases, a wide range of amyloid proteins or peptides such as amyloid-beta and α-synuclein fail to keep native functional conformations, followed by misfolding and self-assembling into a diverse array of aggregates. The aggregates further exert toxicity leading to the dysfunction, degeneration and loss of cells in the affected organs. Due to the disordered structure of the amyloid proteins, endogenous molecules, such as lipids, are prone to interact with amyloid proteins at a low concentration and influence amyloid cytotoxicity. The heterogeneity of amyloid proteinscomplicates the understanding of the amyloid cytotoxicity when relying only on conventional bulk and ensemble techniques. As complementary tools, single-molecule techniques (SMTs) provide novel insights into the different subpopulations of a heterogeneous amyloid mixture as well as the cytotoxicity, in particular as involved in lipid membranes. This review focuses on the recent advances of a series of SMTs, including single-molecule fluorescence imaging, single-molecule force spectroscopy and single-nanopore electrical recording, for the understanding of the amyloid molecular mechanism. The working principles, benefits and limitations of each technique are discussed and compared in amyloid protein related studies.. We also discuss why SMTs show great potential and are worthy of further investigation with feasibility studies as diagnostic tools of neurodegenerative diseases and which limitations are to be addressed.

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

We describe the development of a chemical process based on the CuAAC reaction (click chemistry) to ligate DNA strands and produce an unnatural triazole backbone linkage. The chemical reaction is templated by a complementary DNA splint which accelerates the reaction and provides the required specificity. The resultant 1,4-triazole linkage is read through by DNA and RNA polymerases and is biocompatible in bacterial and human cells. This work has implications for the synthesis of chemically modified genes and other large modified DNA and RNA constructs.

Protein complexes form the critical foundation for a wide range of biological process, however understanding the intricate details of their activities is often challenging. In this review we describe how mass spectrometry (MS) plays a key role in the analysis of protein assemblies and the cellular pathways which they are involved in. Specifically, we discuss how the versatility of mass spectrometric approaches provides unprecedented information on multiple levels. We demonstrate this on the ubiquitin-proteasome proteolytic pathway, a process that is responsible for protein turnover. We follow the various steps of this degradation route and illustrate the different MS workflows that were applied for elucidating molecular information. Overall, this review aims to stimulate the integrated use of multiple mass spectrometry approaches for analyzing complex biological systems.