What is motion? Recent advances in the study of molecular movement patterns of the peptidoglycan synthesis machines

How proteins move through space and time is a fundamental question in biology. While great strides have been made towards a mechanistic understanding of protein movement, many questions remain. We discuss the biological implications of motion in the context of the peptidoglycan (PG) synthesis machines. We review systems in several bacteria, including Escherichia coli , Bacillus subtilis , and Streptococcus pneumoniae , and present a comprehensive view of our current knowledge regarding movement dynamics. Discrepancies are also addressed since “one size does not fit all”. For bacteria to divide, new PG is synthesized and incorporated into the growing cell wall by complex multi-protein nanomachines consisting of PG synthases (transglycosylases [TG] and/or transpeptidases [TP]) as well as a variety of regulators and cytoskeletal factors. Advances in imaging capabilities and labeling methods have revealed that these machines are not static but rather circumferentially transit the cell via directed motion perpendicular to the long axis of model rod-shaped bacteria such as E. coli and B. subtilis . The enzymatic activity of the TG:TPs drives motion in some species, while motion is mediated by FtsZ treadmilling in others. In addition, both directed and diffusive motion of the PG synthases has been observed using single particle tracking technology. Here, we examine the biological role of diffusion regarding transit. Lastly, findings regarding the monofunctional transglycosylases (RodA and FtsW) as well as the Class A PG synthases are discussed. This minireview serves to showcase recent advances, broach mechanistic unknowns, and stimulate future areas of study.

Tachycardia and hypertension enhance tracer efflux from the spinal cord

Background Disruption of cerebrospinal fluid (CSF)/interstitial fluid (ISF) exchange in the spinal cord is likely to contribute to central nervous system (CNS) diseases that involve abnormal fluid accumulation, including spinal cord oedema and syringomyelia. However, the physiological factors that govern fluid transport in the spinal cord are poorly understood. The aims of this study were to determine the effects of cardiac pulsations and respiration on tracer signal increase, indicative of molecular movement following infusion into the spinal cord grey or white matter. Methods In Sprague Dawley rats, physiological parameters were manipulated such that the effects of spontaneous breathing (generating alternating positive and negative intrathoracic pressures), mechanical ventilation (positive intrathoracic pressure only), tachycardia (heart atrial pacing), as well as hypertension (pharmacologically induced) were separately studied. Since fluid outflow from the spinal cord cannot be directly measured, we assessed the molecular movement of fluorescent ovalbumin (AFO-647), visualised by an increase in tracer signal, following injection into the cervicothoracic spinal grey or white matter. Results Tachycardia and hypertension increased AFO-647 tracer efflux, while the concomitant negative and positive intrathoracic pressures generated during spontaneous breathing did not when compared to the positive-pressure ventilated controls. Following AFO-647 tracer injection into the spinal grey matter, increasing blood pressure and heart rate resulted in increased tracer movement away from the injection site compared to the hypotensive, bradycardic animals (hypertension: p = 0.05, tachycardia: p < 0.0001). Similarly, hypertension and tachycardia produced greater movement of AFO-647 tracer longitudinally along the spinal cord following injection into the spinal white matter (p < 0.0001 and p = 0.002, respectively). Tracer efflux was strongly associated with all blood vessel types. Conclusions Arterial pulsations have profound effects on spinal cord interstitial fluid homeostasis, generating greater tracer efflux than intrathoracic pressure changes that occur over the respiratory cycle, demonstrated by increased craniocaudal CSF tracer movement in the spinal cord parenchyma.

Microseepage through Evaporite Sequences – a Gulf of Suez Example

Salt is one of the most effective agents for trapping oil and gas. As a ductile material it can move and deform surrounding sediments and create traps. However, effective sealing of reservoirs for movement of hydrocarbons along breaching faults or fracture swarms (i.e. macroseepage) is a completely different mechanism than the molecular movement of hydrocarbons through grain boundaries and microfractures as found in microseepage. Forum Exploration chose to evaluate the applicability of passive surface geochemistry for mapping hydrocarbons in their onshore West Gebel El Zeit lease due to difficulties in seismic imaging through salt and anhydrites sequences. Two economic producing wells had been drilled in the lease, but due to compartmentalization and complexity in the area, three dry wells had also been drilled. Target formations included the Kareem Formation at ∼2,700 m and the Rudeis Formation at ∼3,000 m.The geochemical survey encompassed 100 passive geochemical modules. Passive samplers were also deployed around two producing wells and one dry well. Calibration data generated positive thermogenic signatures around the two producing wells in contrast to the background or baseline signature developed around the dry well. The Rudeis Formation calibration signature ranged from ∼nC5 - ∼nC9 while the Kareem Formation calibration signature ranged from ∼nC6 – nC12. This suggested the Rudeis calibration signature was lighter than the Kareem. This correlated with independent API gravity testing on produced oil samples (41o API gravity oil for the Rudeis, 35o API gravity oil for the Kareem).A post-survey well, Fh85-8, was drilled based on combined geochemical and seismic data results. The well was an oil discovery, with initial production of 800 BOPD. The evidence presented in this Gulf of Suez example shows that microseepage can occur through salt sequences. As such, ultrasensitive passive surface geochemical surveys provide a powerful tool for derisking salt plays.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Describing the movement of molecules in reduced-dimension models

When addressing spatial biological questions using mathematical models, symmetries within the system are often exploited to simplify the problem by reducing its physical dimension. In a reduced-dimension model molecular movement is restricted to the reduced dimension, changing the nature of molecular movement. This change in molecular movement can lead to quantitatively and even qualitatively different results in the full and reduced systems. Within this manuscript we discuss the condition under which restricted molecular movement in reduced-dimension models accurately approximates molecular movement in the full system. For those systems which do not satisfy the condition, we present a general method for approximating unrestricted molecular movement in reduced-dimension models. We will derive a mathematically robust, finite difference method for solving the 2D diffusion equation within a 1D reduced-dimension model. The methods described here can be used to improve the accuracy of many reduced-dimension models while retaining benefits of system simplification.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP—Pi form stabilizes ribosomal subunit rotation and induces twisting of the sarcin-ricin loop of the 23S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a cascade of rearrangements and a large-scale rotation of EF-G that exerts force on the ribosome and ultimately drives tRNA movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Effects of fatty acid from deep-sea microorganisms on lipid bilayer membrane fluidity under high pressure: comparison of branched-chain and polyunsaturated fatty acid

In this study, the effects of unsaturated and saturated branchedchain fatty acids in biomembranes of microorganisms living under high temperature and high pressure on the fluidity of biomembranes were investigated by time-resolved fluorescence anisotropy measurements. First, the relationship between the order parameter S and the rotational diffusion coefficient Dw, which can be calculated from the fluorescence anisotropy measurements, and the motion of lipid molecules was investigated using lipids with three different structures, and it was found that the former was related to the spacing of lipid molecules and the latter to the motion of lipid molecules. Next, we investigated the S and Dw values of lipid bilayer membrane containing the saturated branched-chain fatty acid 12-methyltridecanoic acid (12-MTA) and the polyunsaturated fatty acid (PUFA) cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). The results showed that 12-MTA increased the S value and decreased the Dw value. On the other hand, DHA tended to reduce the S value and increase the Dw value, albeit slightly. These results mean that 12-MTA narrows the molecular spacing of lipids and inhibits lipid molecular movement, while DHA tends to widen the molecular spacing of lipids and promote lipid molecular movement.

Control of long-distance motion of single molecules on a surface

Spatial control over molecular movement is typically limited because motion at the atomic scale follows stochastic processes. We used scanning tunneling microscopy to bring single molecules into a stable orientation of high translational mobility where they moved along precisely defined tracks. Single dibromoterfluorene molecules moved over large distances of 150 nanometers with extremely high spatial precision of 0.1 angstrom across a silver (111) surface. The electrostatic nature of the effect enabled the selective application of repulsive and attractive forces to send or receive single molecules. The high control allows us to precisely move an individual and specific molecular entity between two separate probes, opening avenues for velocity measurements and thus energy dissipation studies of single molecules in real time during diffusion and collision.