Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Multimodal fast optical interrogation of neural circuitry

The widespread use of molecular machines in biology has long suggested that great rewards could come from bridging the gap between synthetic molecular systems and the machines of the macroscopic world. In the last two decades, it has proved possible to design synthetic molecular systems with architectures where triggered large amplitude positional changes of submolecular components occur. Perhaps the best way to appreciate the technological potential of controlled molecular-level motion is to recognize that nanomotors and molecular-level machines lie at the heart of every significant biological process. Over billions of years of evolution, nature has not repeatedly chosen this solution for performing complex tasks without good reason. When mankind learns how to build artificial structures that can control and exploit molecular level motion and interface their effects directly with other molecular-level substructures and the outside world, it will potentially impact on every aspect of functional molecule and materials design. An improved understanding of physics and biology will surely follow.

The first steps on the long path to the invention of artificial molecular machines were arguably taken in 1827 when the Scottish botanist Robert Brown observed the haphazard motion of tiny particles under his microscope.1,2 The explanation for Brownian motion, that it is caused by bombardment of the particles by molecules as a consequence of the kinetic theory of matter, was later provided by Einstein, followed by experimental verification by Perrin.3,4 The random thermal motion of molecules and its implications for the laws of thermodynamics in turn inspired Gedankenexperiments (“thought experiments”) that explored the interplay (and apparent paradoxes) of Brownian motion and the Second Law of Thermodynamics. Richard Feynman’s famous 1959 lecture “There’s plenty of room at the bottom” outlined some of the promise that manmade molecular machines might hold.5,6 However, Feynman’s talk came at a time before chemists had the necessary synthetic and analytical tools to make molecular machines. While interest among synthetic chemists began to grow in the 1970s and 1980s, progress accelerated in the 1990s, particularly with the invention of methods to make mechanically interlocked molecular systems (catenanes and rotaxanes) and control and switch the relative positions of their components.7−24

Here, we review triggered large-amplitude motions in molecular structures and the changes in properties these can produce. We concentrate on conformational and configurational changes in wholly covalently bonded molecules and on catenanes and rotaxanes in which switching is brought about by various stimuli (light, electrochemistry, pH, heat, solvent polarity, cation or anion binding, allosteric effects, temperature, reversible covalent bond formation, etc.). Finally, we discuss the latest generations of sophisticated synthetic molecular machine systems in which the controlled motion of subcomponents is used to perform complex tasks, paving the way to applications and the realization of a new era of “molecular nanotechnology”.

1.1. The Language Used To Describe Molecular Machines
Terminology needs to be properly and appropriately defined and these meanings used consistently to effectively convey scientific concepts. Nowhere is the need for accurate scientific language more apparent than in the field of molecular machines. Much of the terminology used to describe molecular-level machines has its origins in observations made by biologists and physicists, and their findings and descriptions have often been misinterpreted and misunderstood by chemists. In 2007 we formalized definitions of some common terms used in the field (e.g., “machine”, “switch”, “motor”, “ratchet”, etc.) so that chemists could use them in a manner consistent with the meanings understood by biologists and physicists who study molecular-level machines.14

The word “machine” implies a mechanical movement that accomplishes a useful task. This Review concentrates on systems where a stimulus triggers the controlled, relatively large amplitude (or directional) motion of one molecular or submolecular component relative to another that can potentially result in a net task being performed. Molecular machines can be further categorized into various classes such as “motors” and “switches” whose behavior differs significantly.14 For example, in a rotaxane-based “switch”, the change in position of a macrocycle on the thread of the rotaxane influences the system only as a function of state. Returning the components of a molecular switch to their original position undoes any work done, and so a switch cannot be used repetitively and progressively to do work. A “motor”, on the other hand, influences a system as a function of trajectory, meaning that when the components of a molecular motor return to their original positions, for example, after a 360° directional rotation, any work that has been done is not undone unless the motor is subsequently rotated by 360° in the reverse direction. This difference in behavior is significant; no “switch-based” molecular machine can be used to progressively perform work in the way that biological motors can, such as those from the kinesin, myosin, and dynein superfamilies, unless the switch is part of a larger ratchet mechanism.14

Artificial Molecular Machines

The review covers the knowledge on photoremovable protecting
groups and includes all relevant chromophores studied in the
time period of 2000–2012; the most relevant earlier works are
also discussed.

/pdf/photoremovable-protecting-groups-in-chemistry-and-biology-2n33qs6p7b.pdf

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

Fluorescence imaging techniques have been widely used to visualize biological molecules and phenomena. In particular, several studies on the development of small-molecule fluorescent probes have been carried out, because their fluorescence properties can be easily tuned by synthetic chemical modification. For this reason, various fluorescent probes have been developed for targeting biological components, such as proteins, peptides, amino acids, and ions, to the interior and exterior of cells. In this review, we cover advances in the development of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based fluorescent probes for biological studies over the past decade.

BODIPY-based probes for the fluorescence imaging of biomolecules in living cells

Pharmacotherapy is often severely hindered by issues related to poor drug selectivity, including side effects, environmental toxicity, and the emergence of resistance. Lack of selectivity is caused by the inability to control drug activity in time and space. Photopharmacology aims at solving this issue by incorporating photoswitchable groups into the molecular structure of bioactive compounds. These switching units allow for the use of light as an external control element for pharmacological activity, which can be delivered with very high spatiotemporal precision. This Perspective presents the reader with the current state and outlook on photopharmacology. In particular, the principles behind photoregulation of bioactivity, the challenges of molecular design, and the possible therapeutic scenarios are discussed.

/pdf/photopharmacology-beyond-proof-of-principle-apdgap8fji.pdf

Photopharmacology: Beyond Proof of Principle

Spatial and temporal control over chemical and biological processes plays a key role in life, where the whole is often much more than the sum of its parts. Quite trivially, the molecules of a cell do not form a living system if they are only arranged in a random fashion. If we want to understand these relationships and especially the problems arising from malfunction, tools are necessary that allow us to design sophisticated experiments that address these questions. Highly valuable in this respect are external triggers that enable us to precisely determine where, when, and to what extent a process is started or stopped. Light is an ideal external trigger: It is highly selective and if applied correctly also harmless. It can be generated and manipulated with well-established techniques, and many ways exist to apply light to living systems--from cells to higher organisms. This Review will focus on developments over the last six years and includes discussions on the underlying technologies as well as their applications.

Light-Controlled Tools

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Light‐Controlled Tools

https://pubs.rsc.org/en/content/articlepdf/2015/np/c5np00025d

Structural aspects of phenylglycines, their biosynthesis and occurrence in peptide natural products

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.

Clara Brieke

Papers

Light-Controlled Tools

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Light‐Controlled Tools

Structural aspects of phenylglycines, their biosynthesis and occurrence in peptide natural products

The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase