Single molecule kinetics of bacteriorhodopsin by HS-AFM

Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.

Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase

Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion. Thus, the enzyme’s catalytic cycle may have to be turned by 180 degrees.

A hold-and-feed mechanism drives directional DNA loop extrusion by condensin

SMC protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop in two separate chambers. Single-molecule and cryo-electron microscopy provide evidence for a power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon ATP binding, while the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of 'motor' and 'anchor' chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the course of the SMC reaction cycle determines the directionality of DNA loop extrusion.

Memory, switches, and logic gates through bistability in chemically fueled crystals

The ability to store information in chemical reaction networks is essential for evolution, calculations, and, more generally, for the complex behavior, we associate with life. In biology, cellular memory is regulated through transcriptional states that are bistable, i.e., a state that can either be on or off and can be flipped from one to another through a transient signal. Such memory circuits have been realized synthetically through the rewiring of genetic systems in vivo or through the rational design of reaction networks based on DNA and highly evolved enzymes in vitro. Completely bottom-up analogs based on small molecules are rare and hard to design and thus represent a challenge for systems chemistry. In this work, we show that bistability can be designed from an extremely simple non-equilibrium reaction cycle that is coupled to crystallization. The crystals exert the necessary feedback on the reaction cycle required for the bistability resulting in an on-state with assemblies and an off-state without. We can switch the state on and off, such that each state represents volatile memory that can be stored in continuously stirred tank reactors indefinitely despite the fact that molecules are turned over on a minute-timescale. We showcase the system’s abilities by creating a matrix display that can store images and by performing Boolean logic by coupling several switches together.

Space and Time Crystal Engineering in Developing Futuristic Chemical Technology

In the coming years, multipurpose catalysts for delivering different products under the same chemical condition will be required for developing smart devices for industrial or household use. In order to design such multipurpose devices with two or more specific roles, we need to incorporate a few independent but externally controllable catalytically active centers. Through space crystal engineering, such an externally controllable multipurpose MOF-based photocatalyst could be designed. In a chemical system, a few mutually independent secondary reaction cycles nested within the principal reaction cycle can be activated externally to yield different competitive products. Each reaction cycle can be converted into a time crystal, where the time consuming each reaction step could be converted as an event and all the reaction steps or events could be connected by a circle to build a time crystal. For fractal reaction cycles, a time polycrystal can be generated. By activating a certain fractal event based nested time crystal branch, we can select one of the desired competitive products according to our needs. This viewpoint intends to bring together the ideas of (spatial) crystal engineering and time crystal engineering in order to make use of the time–space arrangement in reaction–catalysis systems and introduce new aspects to futuristic chemical engineering technology.

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.