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.

When the iconic DNA genetic code is expressed in terms of energy differentials, one observes that information embedded in chemical sequences, including some biological outcomes, correlate with distinctive free energy profiles. Specifically, we find correlations between codon usage and codon free energy, suggestive of a thermodynamic selection for codon usage. We also find correlations between what are considered ancient amino acids and high codon free energy values. Such correlations may be reflective of the sequence-based genetic code fundamentally mapping as an energy code. In such a perspective, one can envision the genetic code as composed of interlocking thermodynamic cycles that allow codons to ‘evolve’ from each other through a series of sequential transitions and transversions, which are influenced by an energy landscape modulated by both thermodynamic and kinetic factors. As such, early evolution of the genetic code may have been driven, in part, by differential energetics, as opposed exclusively by the functionality of any gene product. In such a scenario, evolutionary pressures can, in part, derive from the optimization of biophysical properties (e.g. relative stabilities and relative rates), in addition to the classic perspective of being driven by a phenotypical adaptive advantage (natural selection). Such differential energy mapping of the genetic code, as well as larger genomic domains, may reflect an energetically resolved and evolved genomic landscape, consistent with a type of differential, energy-driven ‘molecular Darwinism’. It should not be surprising that evolution of the code was influenced by differential energetics, as thermodynamics is the most general and universal branch of science that operates over all time and length scales.

Flagellar dyneins are the molecular motors responsible for producing the propagating bending motions of cilia and flagella. They are located within a densely packed and highly organised super-macromolecular cytoskeletal structure known as the axoneme. Using the mesoscale simulation technique Fluctuating Finite Element Analysis (FFEA), which represents proteins as viscoelastic continuum objects subject to explicit thermal noise, we have quantified the constraints on the range of molecular conformations that can be explored by dynein-c within the crowded architecture of the axoneme. We subsequently assess the influence of crowding on the 3D exploration of microtubule-binding sites, and specifically on the axial step length. Our calculations combine experimental information on the shape, flexibility and environment of dynein-c from three distinct sources; negative stain electron microscopy, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). Our FFEA simulations show that the super-macromolecular organisation of multiple protein complexes into higher-order structures can have a significant influence on the effective flexibility of the individual molecular components, and may, therefore, play an important role in the physical mechanisms underlying their biological function.

DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

Darwin's theory of evolution emphasized that positive selection of functional proficiency provides the fitness that ultimately determines the structure of life, a view that has dominated biochemical thinking of enzymes as perfectly optimized for their specific functions. The 20th-century modern synthesis, structural biology, and the central dogma explained the machinery of evolution, and nearly neutral theory explained how selection competes with random fixation dynamics that produce molecular clocks essential e.g. for dating evolutionary histories. However, quantitative proteomics revealed that selection pressures not relating to optimal function play much larger roles than previously thought, acting perhaps most importantly via protein expression levels. This paper first summarizes recent progress in the 21st century toward recovering this universal selection pressure. Then, the paper argues that proteome cost minimization is the dominant, underlying ‘non-function’ selection pressure controlling most of the evolution of already functionally adapted living systems. A theory of proteome cost minimization is described and argued to have consequences for understanding evolutionary trade-offs, aging, cancer, and neurodegenerative protein-misfolding diseases.

The behavior of molecules confined to small spaces is fascinating chemistry and lies at the heart of signaling processes in biology. Our approach to confinement is through reversible encapsulation of small molecules in synthetic containers. We show that confinement leads to amplified reactivities in bimolecular reactions, stabilization of otherwise reactive species, and limitation in motions that create new stereochemical arrangements. The isolation of molecules from solvent makes for manageable computations and has stimulated theorist to examine reaction details in the limited space. Transition states for reactions and rearrangements can be calculated, the effects of (de)solvation can be evaluated and the magnetic properties of the containers can be compared with experimental observations. Finally, we outline several potential applications, including entanglement chemistry and the use of isomers in data storage.

Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases. We discuss in particular models based on the transgenic expression of amyloid-β (Aβ) and tau in AD, and α-synuclein in PD. Because biophysical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro, we expect that the further development of these methods to probe directly the corresponding mechanisms in vivo will open effective routes for diagnostic and therapeutic interventions.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4–TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide–TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.

Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.

Among the unsolved mysteries of modern biology is the nature of a lining of blood vessels called the ‘endothelial surface layer’ or ESL. In venous micro-vessels, it is half a micron in thickness. The ESL is 10 times thicker than the endothelial glycocalyx (eGC) at its base, has been presumed to be comprised mainly of water, yet is rigid enough to exclude red blood cells. How is this possible? Developments in physical chemistry suggest that the venous ESL is actually comprised of nanobubbles of CO2, generated from tissue metabolism, in a foam nucleated in the eGC. For arteries, the ESL is dominated by nanobubbles of O2 and N2 from inspired air. The bubbles of the foam are separated and stabilized by thin layers of serum electrolyte and proteins, and a palisade of charged polymer strands of the eGC. The ESL seems to be a respiratory organ contiguous with the flowing blood, an extension of, and a ‘lung’ in miniature. This interpretation may have far-reaching consequences for physiology.

Few proteins have come under such intense scrutiny as superoxide dismutase-1 (SOD1). For almost a century, scientists have dissected its form, function and then later its malfunction in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We now know SOD1 is a zinc and copper metalloenzyme that clears superoxide as part of our antioxidant defence and respiratory regulation systems. The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in the sod1 gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophysical studies examined the characteristics of mutant SOD1 in great detail. We now recognise how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insoluble inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wild-type SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighbouring cells. This raises the possibility of intervention after the initial disease presentation. Several small-molecule and biologic-based strategies have been devised which directly target the SOD1 molecule to change the behaviour thought to be responsible for ALS. Here we provide a comprehensive review of the many biophysical advances that sculpted our view of SOD1 biology and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.

The strong, long-range electrostatic forces described by Coulomb's law disappear for ions in water, and the behavior of these ions is instead controlled by their water affinity – a weak, short-range force which arises from their charge density. This was established experimentally in the mid-1980s by size-exclusion chromatography on carefully calibrated Sephadex® G-10 (which measures the effective volume and thus the water affinity of an ion) and by neutron diffraction with isotopic substitution (which measures the density and orientation of water molecules near the diffracting ion and thus its water affinity). These conclusions have been confirmed more recently by molecular dynamics simulations, which explicitly model each individual water molecule. This surprising change in force regime occurs because the oppositely charged ions in aqueous salt solutions exist functionally as ion pairs (separated by 0, 1 or 2 water molecules) as has now been shown by dielectric relaxation spectroscopy; this cancels out the strong long-range electrostatic forces and allows the weak, short-range water affinity effects to come to the fore. This microscopic structure of aqueous salt solutions is not captured by models utilizing a macroscopic dielectric constant. Additionally, the Law of Matching Water Affinity, first described in 1997 and 2004, establishes that contact ion pair formation is controlled by water affinity and is a major determinant of the solubility of charged species since only a net neutral species can change phases.

Integrins are large heterodimeric type 1 membrane proteins expressed in all nucleated mammalian cells. Eighteen α-chains and eight β-chains can combine to form 24 different integrins. They are cell adhesion proteins, which bind to a large variety of cellular and extracellular ligands. Integrins are required for cell migration, hemostasis, translocation of cells out from the blood stream and further movement into tissues, but also for the immune response and tissue morphogenesis. Importantly, integrins are not usually active as such, but need activation to become adhesive. Integrins are activated by outside-in activation through integrin ligand binding, or by inside-out activation through intracellular signaling. An important question is how integrin activity is regulated, and this topic has recently drawn much attention. Changes in integrin affinity for ligand binding are due to allosteric structural alterations, but equally important are avidity changes due to integrin clustering in the plane of the plasma membrane. Recent studies have partially solved how integrin cell surface structures change during activation. The integrin cytoplasmic domains are relatively short, but by interacting with a variety of cytoplasmic proteins in a regulated manner, the integrins acquire a number of properties important not only for cell adhesion and movement, but also for cellular signaling. Recent work has shown that specific integrin phosphorylations play pivotal roles in the regulation of integrin activity. Our purpose in this review is to integrate the present knowledge to enable an understanding of how cell adhesion is dynamically regulated.

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.

Fluorescence turn-on aptamers, in vitro evolved RNA molecules that bind conditional fluorophores and activate their fluorescence, have emerged as RNA counterparts of the fluorescent proteins. Turn-on aptamers have been selected to bind diverse fluorophores, and they achieve varying degrees of specificity and affinity. These RNA–fluorophore complexes, many of which exceed the brightness of green fluorescent protein and their variants, can be used as tags for visualizing RNA localization and transport in live cells. Structure determination of several fluorescent RNAs revealed that they have diverse, unrelated overall architectures. As most of these RNAs activate the fluorescence of their ligands by restraining their photoexcited states into a planar conformation, their fluorophore binding sites have in common a planar arrangement of several nucleobases, most commonly a G-quartet. Nonetheless, each turn-on aptamer has developed idiosyncratic structural solutions to achieve specificity and efficient fluorescence turn-on. The combined structural diversity of fluorophores and turn-on RNA aptamers has already produced combinations that cover the visual spectrum. Further molecular evolution and structure-guided engineering is likely to produce fluorescent tags custom-tailored to specific applications.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotechnology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstration of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.