Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli

Biotechnology and Biological Sciences Research Council. Grant Number: BB/E009571, Oxford Centre for Integrative Systems Biology (OCISB), Engineering and Physical Science Research Council, Royal Society, Hertford College Oxfor

Membranes by the Numbers

Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.Comment: 27 pages, 12 figure

Analysis of noise in differential and ratiometric biosensing systems

This paper presents formulations to evaluate noise in differential and ratiometric measurements that are often performed in biosensing. These measurements are performed to improve signal to noise ratio of the sensing systems for sensitive detection of dynamic biological processes. The use of these formulations is discussed in the context of the differential intensity surface plasmon resonance (SPR) system that is widely used to characterise molecular interactions on a confined axial scale. Previous studies provide qualitative descriptions of the noise performance of such systems but lack rigorous characterisation. Here we present analytical expressions for quantitative evaluation of the noise in differential and ratiometric measurements by applying the rules of arithmetic operations on random variables. Such formulations provide the means for evaluating the signal to noise ratio of such systems. We present how correlated noise can be removed by performing differential or ratiometric processing. Applying these formulations, we also show how the sensitivity of the differential intensity SPR system changes during the experiment

Highly sensitive multipoint real-time kinetic detection of Surface Plasmon bioanalytes with custom CMOS cameras

Phase sensitive Surface Plasmon Resonance (SPR) techniques are a popular means of characterizing biomolecular interactions. However, limitations due to the narrow dynamic range and difficulty in adapting the method for multi-point sensing have restricted its range of applications. This paper presents a compact phase sensitive SPR technology using a custom CMOS camera. The system is exceptionally versatile enabling one to trade dynamic range for sensitivity without altering the optical system. We present results showing sensitivity over the array of better than 10−6 Refractive Index Units (RIU) over a refractive index range of 2×10−2 RIU, with peak sensitivity of 3×10−7 RIU at the center of this range. We also explain how simply altering the settings of polarization components can give sensitivity on the order of 10−8 RIU albeit at the cost of lower dynamic range. The consistent response of the custom CMOS camera in the system also allowed us to demonstrate precise quantitative detection of two Fibrinogen antibody–protein binding sites. Moreover, we use the system to determine reaction kinetics and argue how the multipoint detection gives useful insight into the molecular binding mechanisms

Unique Properties of Eukaryote-Type Actin and Profilin Horizontally Transferred to Cyanobacteria

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 µm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 µm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism

Heterogeneous localisation of membrane proteins in Staphylococcus aureus

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation