The biological energy cycle of our planet is driven by photosynthesis whereby sunlight is absorbed by chlorophyll and other accessory pigments. The excitation energy is then efficiently transferred to a reaction centre where charge separation occurs in a few picoseconds. In the case of photosystem II (PSII), the energy of the charge transfer state is used to split water into oxygen and reducing equivalents. This is accomplished by the relatively low energy content of four photons of visible light. PSII is a large multi-subunit membrane protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Four high energy electrons, together with four protons (4H+), are used to reduce plastoquinone (PQ), the terminal electron acceptor of PSII, to plastoquinol (PQH2). PQH2 passes its reducing equivalents to an electron transfer chain which feeds into photosystem I (PSI) where they gain additional reducing potential from a second light reaction which is necessary to drive CO2 reduction. The catalytic centre of PSII consists of a cluster of four Mn ions and a Ca2+ linked by oxo bonds. In addition, there are seven amino acid ligands. In this Article, I discuss the structure of this metal cluster, its stability and the probability that an acid-base (nucleophilic-electrophilic) mechanism catalyses the water splitting reaction on the surface of the metal-cluster. Evidence for this mechanism is presented from studies on water splitting catalysts consisting of organo-complexes of ruthenium and manganese and also by comparison with the enzymology of carbon monoxide dehydrogenase (CODH). Finally the relevance of our understanding of PSII is discussed in terms of artificial photosynthesis with emphasis on inorganic water splitting catalysts as oxygen generating photoelectrodes.

Ribosomes are remarkable ribonucleoprotein complexes that are responsible for protein synthesis in all forms of life. They polymerize polypeptide chains programmed by nucleotide sequences in messenger RNA in a mechanism mediated by transfer RNA. One of the most challenging problems in the ribosome field is to understand the mechanism of coupled translocation of mRNA and tRNA during the elongation phase of protein synthesis. In recent years, the results of structural, biophysical and biochemical studies have provided extensive evidence that translocation is based on the structural dynamics of the ribosome itself. Detailed structural analysis has shown that ribosome dynamics, like aminoacyl-tRNA selection and catalysis of peptide bond formation, is made possible by the properties of ribosomal RNA.

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair)−1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3′−3′ strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a ‘disproportionation’ into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

Bacterial membranes represent an attractive target for the design of new antibiotics to combat widespread bacterial resistance to traditional inhibitor-based antibiotics. Understanding how antimicrobial peptides (AMPs) and other membrane-active agents attack membranes could facilitate the design of new, effective antimicrobials. AMPs, which are small, gene-encoded host defense proteins, offer a promising basis for the study of membrane-active antimicrobial agents. These peptides are cationic and amphipathic, spontaneously binding to bacterial membranes and inducing transmembrane permeability to small molecules. Yet there are often confusions surrounding the details of the molecular mechanisms of AMPs. Following the doctrine of structure–function relationship, AMPs are often viewed as the molecular scaffolding of pores in membranes. Instead we believe that the full mechanism of AMPs is understandable if we consider the interactions of AMPs with the whole membrane domain, where interactions induce structural transformations of the entire membrane, rather than forming localized molecular structures. We believe that it is necessary to consider the entire soft matter peptide-membrane system as it evolves through several distinct states. Accordingly, we have developed experimental techniques to investigate the state and structure of the membrane as a function of the bound peptide to lipid ratio, exactly as AMPs in solution progressively bind to the membrane and induce structural changes to the entire system. The results from these studies suggest that global interactions of AMPs with the membrane domain are of fundamental importance to understanding the antimicrobial mechanisms of AMPs.

The kinetics of biochemical and biophysical events determined the course of life processes and attracted considerable interest and research. For example, modeling of biological networks and cellular responses relies on the availability of information on rate coefficients. Atomically detailed simulations hold the promise of supplementing experimental data to obtain a more complete kinetic picture. However, simulations at biological time scales are challenging. Typical computer resources are insufficient to provide the ensemble of trajectories at the correct length that is required for straightforward calculations of time scales. In the last years, new technologies emerged that make atomically detailed simulations of rate coefficients possible. Instead of computing complete trajectories from reactants to products, these approaches launch a large number of short trajectories at different positions. Since the trajectories are short, they are computed trivially in parallel on modern computer architecture. The starting and termination positions of the short trajectories are chosen, following statistical mechanics theory, to enhance efficiency. These trajectories are analyzed. The analysis produces accurate estimates of time scales as long as hours. The theory of Milestoning that exploits the use of short trajectories is discussed, and several applications are described.

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.

The nucleosome serves as a general gene repressor, preventing all initiation of transcription except that which is brought about by specific positive regulatory mechanisms. The positive mechanisms begin with chromatin-remodeling by complexes that slide, disrupt, or otherwise alter the structure and organization of nucleosomes. RSC in yeast and its counterpart PBAF in human cells are the major remodeling complexes for transcription. RSC creates a nucleosome-free region in front of a gene, flanked by strongly positioned +1 and −1 nucleosomes, with the transcription start site typically 10–15 bp inside the border of the +1 nucleosome. RSC also binds stably to nucleosomes harboring regulatory elements and to +1 nucleosomes, perturbing their structures in a manner that partially exposes their DNA sequences. The cryo-electron microscope structure of a RSC–nucleosome complex reveals such a structural perturbation, with the DNA largely unwrapped from the nucleosome and likely interacting with a positively charged surface of RSC. Such unwrapping both exposes the DNA and enables its translocation across the histone octamer of the nucleosome by an ATP-dependent activity of RSC. Genetic studies have revealed additional roles of RSC in DNA repair, chromosome segregation, and other chromosomal DNA transactions. These functions of RSC likely involve the same fundamental activities, DNA unwrapping and DNA translocation.

The emergence of functional cooperation between the three main classes of biomolecules – nucleic acids, peptides and lipids – defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an ‘RNA world’, notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pKa values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.

Amyloid formation of the synaptic brain protein α-synuclein (αS) is related to degeneration of dopaminergic neurons in Parkinson's disease patients. αS is thought to function in vesicle transport and fusion and it binds strongly to negatively charged vesicles in vitro. Here we combined circular dichroism, fluorescence and imaging methods in vitro to characterize the interaction of αS with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), sodium salt) and the consequences of such interactions on αS amyloid formation. We found that lipid head-group chemistry modulates αS interactions and also affects amyloid fiber formation. During the course of the experiments, we made the unexpected discovery that pre-formed αS oligomers, typically present in a small amount in the αS starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining αS monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden αS aggregation pathway may be of importance in vivo.

Kinetics of acid-induced chlorophyll demetallation was recorded in microdroplets by fusing a stream of microdroplets containing 40 µM chlorophyll a or b dissolved in methanol with a stream of aqueous microdroplets containing 35 mM hydrochloric acid (pH = 1·46). The kinetics of the demetallation of chlorophyll in the fused microdroplets (14 ± 6 µm diameter; 84 ± 18 m s−1 velocity) was recorded by controlling the traveling distance of the fused microdroplets between the fusion region and the inlet of a mass spectrometer. The rate of acid-induced chlorophyll demetallation was about 960 ± 120 times faster in the charged microdroplets compared with that reported in bulk solution. If no voltage was applied to the sprayed microdroplets, then the acceleration factor was about 580 ± 90, suggesting that the applied voltage is not a major factor determining the acceleration. Chlorophyll a was more rapidly demetallated than chlorophyll b by a factor of ~26 in bulk solution and ~5 in charged microdroplets. The demetallation kinetics was second order in the H+ concentration, but the acceleration factor of microdroplets compared with bulk solution appeared to be unchanged in going from pH = 1·3 to 7·0. The water:methanol ratio of the fused microdroplets was varied from 7:3 to 3:7 causing an increase in the reaction rate of chlorophyll a demetallation by 20%. This observation demonstrates that the solvent composition, which has different evaporation rates, does not significantly affect the acceleration. We believe that a major portion of the acceleration can be attributed to confinement effects involving surface reactions rather than either to evaporation of solvents or to the introduction of charges to the microdroplets.

In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.

In 1912, Max von Laue and collaborators first observed diffraction spots from a millimeter-sized crystal of copper sulfate using an X-ray tube. Crystallography was born of this experiment, and since then, diffraction by both X-rays and electrons has revealed a myriad of inorganic and organic structures, including structures of complex protein assemblies. Advancements in X-ray sources have spurred a revolution in structure determination, facilitated by the development of new methods. This review explores some of the frontier methods that are shaping the future of X-ray diffraction, including coherent diffractive imaging, serial femtosecond X-ray crystallography and small-angle X-ray scattering. Collectively, these methods expand the current limits of structure determination in biological systems across multiple length and time scales.

Live-cell and live-tissue imaging using fluorescence optical microscopes presents an inherent trade-off between image quality and photodamage. Spatially-controlled illumination microscopy (SCIM) aims to strike the right balance between obtaining good image quality and minimizing the risk of photodamage. In traditional imaging, illumination is performed with a spatially-uniform light dose resulting in spatially-variable detected signals. SCIM adopts an alternative imaging approach where illumination is performed with a spatially-variable light dose resulting in spatially-uniform detected signals. The actual image information of the biological specimen in SCIM is predominantly encoded in the illumination profile. SCIM uses real-time spatial control of illumination in the imaging of fluorescent biological specimens. This alternative imaging paradigm reduces the overall illumination light dose during imaging, which facilitates prolonged imaging of live biological specimens by minimizing photodamage without compromising image quality. Additionally, the dynamic range of a SCIM image is no longer limited by the dynamic range of the detector (or camera), since it employs a uniform detection strategy. The large dynamic range of SCIM is predominantly determined by the illumination profile, and is advantageous for imaging both live and fixed biological specimens. In the present review, the concept and working mechanisms of SCIM are discussed, together with its application in various types of optical microscopes.

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.

Einstein was wrong with his 1927 Solvay Conference claim that quantum mechanics is incomplete and incapable of describing diffraction of single particles. However, the Einstein-Podolsky-Rosen paradox of entangled pairs of particles remains lurking with its ‘spooky action at a distance’. In molecules quantum entanglement can be viewed as basis of both chemical bonding and excitonic states. The latter are important in many biophysical contexts and involve coupling between subsystems in which virtual excitations lead to eigenstates of the total Hamiltonian, but not for the separate subsystems. The author questions whether atomic or photonic systems may be probed to prove that particles or photons may stay entangled over large distances and display the immediate communication with each other that so concerned Einstein. A dissociating hydrogen molecule is taken as a model of a zero-spin entangled system whose angular momenta are in principle possible to probe for this purpose. In practice, however, spins randomize as a result of interactions with surrounding fields and matter. Similarly, no experiment seems yet to provide unambiguous evidence of remaining entanglement between single photons at large separations in absence of mutual interaction, or about immediate (superluminal) communication. This forces us to reflect again on what Einstein really had in mind with the paradox, viz. a probabilistic interpretation of a wave function for an ensemble of identically prepared states, rather than as a statement about single particles. Such a prepared state of many particles would lack properties of quantum entanglement that make it so special, including the uncertainty upon which safe quantum communication is assumed to rest. An example is Zewail's experiment showing visible resonance in the dissociation of a coherently vibrating ensemble of NaI molecules apparently violating the uncertainty principle. Einstein was wrong about diffracting single photons where space-like anti-bunching observations have proven recently their non-local character and how observation in one point can remotely affect the outcome in other points. By contrast, long range photon entanglement with immediate, superluminal response is still an elusive, possibly partly misunderstood issue. The author proposes that photons may entangle over large distances only if some interaction exists via fields that cannot propagate faster than the speed of light. An experiment to settle this ‘interaction hypothesis’ is suggested.

Increasing evidence suggests that most proteins occur and function in complexes rather than as isolated entities when embedded in cellular membranes. Nuclear magnetic resonance (NMR) provides increasing possibilities to study structure, dynamics and assembly of such systems. In our review, we discuss recent methodological progress to study membrane–protein complexes (MPCs) by NMR, starting with expression, isotope-labeling and reconstitution protocols. We review approaches to deal with spectral complexity and limited spectral spectroscopic sensitivity that are usually encountered in NMR-based studies of MPCs. We highlight NMR applications in various classes of MPCs, including G-protein-coupled receptors, ion channels and retinal proteins and extend our discussion to protein–protein complexes that span entire cellular compartments or orchestrate processes such as protein transport across or within membranes. These examples demonstrate the growing potential of NMR-based studies of MPCs to provide critical insight into the energetics of protein–ligand and protein–protein interactions that underlie essential biological functions in cellular membranes.

About 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of reducing equivalents needed to convert carbon dioxide into the organic molecules of life while at the same time produced oxygen to transform our planetary atmosphere from an anaerobic to an aerobic state. The enzyme which facilitates this reaction and therefore underpins virtually all life on our planet is known as Photosystem II (PSII). It is a pigment-binding, multisubunit protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Today we have detailed understanding of the structure and functioning of this key and unique enzyme. The journey to this level of knowledge can be traced back to the discovery of oxygen itself in the 18th-century. Since then there has been a sequence of mile stone discoveries which makes a fascinating story, stretching over 200 years. But it is the last few years that have provided the level of detail necessary to reveal the chemistry of water oxidation and O–O bond formation. In particular, the crystal structure of the isolated PSII enzyme has been reported with ever increasing improvement in resolution. Thus the organisational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino-acid side chains, of which seven provide direct ligands to the metals. The metal cluster is organised as a cubane structure composed of three Mn ions and a Ca2+ linked by oxo-bonds with the fourth Mn ion attached to the cubane. This structure has now been synthesised in a non-protein environment suggesting that it is a totally inorganic precursor for the evolution of the photosynthetic oxygen-evolving complex. In summary, the overall structure of the catalytic site has given a framework on which to build a mechanistic scheme for photosynthetic dioxygen generation and at the same time provide a blue-print and incentive to develop catalysts for artificial photo-electrochemical systems to split water and generate renewable solar fuels.