Thomas L. Rodgers

CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2. DOI: http://dx.doi.org/10.7554/eLife.01213.001

Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

Allostery in bacterial transcription factors arises from changes in global low-frequency protein dynamics. Amino acids that regulate low-frequency dynamics are identified and seen to be evolutionarily conserved.

Allostery without conformation change: modelling protein dynamics at multiple scales

The original ideas of Cooper and Dryden, that allosteric signalling can be induced between distant binding sites on proteins without any change in mean structural conformation, has proved to be a remarkably prescient insight into the rich structure of protein dynamics. It represents an alternative to the celebrated Monod-Wyman-Changeux mechanism and proposes that modulation of the amplitude of thermal fluctuations around a mean structure, rather than shifts in the structure itself, give rise to allostery in ligand binding. In a complementary approach to experiments on real proteins, here we take a theoretical route to identify the necessary structural components of this mechanism. By reviewing and extending an approach that moves from very coarse-grained to more detailed models, we show that, a fundamental requirement for a body supporting fluctuation-induced allostery is a strongly inhomogeneous elastic modulus. This requirement is reflected in many real proteins, where a good approximation of the elastic structure maps strongly coherent domains onto rigid blocks connected by more flexible interface regions.

Dynamic Transmission of Protein Allostery without Structural Change: Spatial Pathways or Global Modes?

We examine the contrast between mechanisms for allosteric signaling that involve structural change, and those that do not, from the perspective of allosteric pathways. In particular we treat in detail the case of fluctuation-allostery by which amplitude modulation of the thermal fluctuations of the elastic normal modes conveys the allosteric signal, and address the question of what an allosteric pathway means in this case. We find that a perturbation theory of thermal elastic solids and nonperturbative approach (by super-coarse-graining elasticity into internal bending modes) have opposite signatures in their structure of correlated pathways. We illustrate the effect from analysis of previous results from GlxR of Corynebacterium glutamicum, an example of the CRP/FNR transcription family of allosteric homodimers. We find that the visibility of both correlated pathways and disconnected sites of correlated motion in this protein suggests that mechanisms of local elastic stretch and bend are recruited for the purpose of creating and controlling allosteric cooperativity.

Global low-frequency motions in protein allostery: CAP as a model system.

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. There is considerable evidence that allosteric cooperativity can be communicated by the modulation of protein dynamics without conformational change. The Catabolite Activator Protein (CAP) of Escherichia coli is an important experimental exemplar for entropically driven allostery. Here we discuss recent experimentally supported theoretical analysis that highlights the role of global low-frequency dynamics in allostery in CAP and identify how allostery arises as a natural consequence of changes in global low-frequency protein fluctuations on ligand binding.

ΔΔPT: a comprehensive toolbox for the analysis of protein motion

BackgroundNormal Mode Analysis is one of the most successful techniques for studying motions in proteins and macromolecules. It can provide information on the mechanism of protein functions, used to aid crystallography and NMR data reconstruction, and calculate protein free energies.ResultsΔΔPT is a toolbox allowing calculation of elastic network models and principle component analysis. It allows the analysis of pdb files or trajectories taken from; Gromacs, Amber, and DL_POLY. As well as calculation of the normal modes it also allows comparison of the modes with experimental protein motion, variation of modes with mutation or ligand binding, and calculation of molecular dynamic entropies.ConclusionsThis toolbox makes the respective tools available to a wide community of potential NMA users, and allows them unrivalled ability to analyse normal modes using a variety of techniques and current software.

Comparison of a networks-of-zones fluid mixing model for a baffled stirred vessel with three-dimensional electrical resistance tomography

Reliable models for the simulation of mixing vessels are important for the understanding of real-life mixing problems. To achieve these models, information about the mixing in the system must be measured to compare with the predicted values. Electrical resistance tomography has the capability to measure spatial and temporal changes within a vessel in three dimensions even in optically inaccessible environments. This paper discusses the creation of a network-of-zones model for the prediction of mixing within a vessel with a Cowles disc-type agitator. Solving of the network-of-zones simplified transport equations for the vessel predicts the concentration distribution of an inert tracer added to the vessel. The change in this distribution with time is calculated and compared with visual inspection of the vessel. The concentration distribution inside the vessel is also measured using electrical resistance tomography and shows good agreement with the predicted values.

An SPR based sensor for allergens detection.

A simple, sensitive and label-free optical sensor method was developed for allergens analysis using α-casein as the biomarker for cows milk detection, to be used directly in final rinse samples of cleaning in place systems (CIP) of food manufacturers. A Surface Plasmon Resonance (SPR) sensor chip consisting of four sensing arrays enabling the measurement of samples and control binding events simultaneously on the sensor surface was employed in this work. SPR offers several advantages in terms of label free detection, real time measurements and superior sensitivity when compared to ELISA based techniques. The gold sensor chip was used to immobilise α-casein-polyclonal antibody using EDC/NHS coupling procedure. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions giving a detection limit of 58ngmL-1 as a direct binding assay. The assay sensitivity can be further improved by using sandwich assay format and amplified with nanoparticles. However, at this stage this is not required as the detection limit achieved exceeded the required allergens detection levels of 2µgmL-1 for α-S1-casein. The sensor demonstrated good selectivity towards the α-casein as the target analyte and adequate recoveries from CIP final rinse wash samples. The sensor would be useful tool for monitoring allergen levels after cleaning procedures, providing additional data that may better inform upon wider food allergen risk management decision(s) that are made by food manufacturer. In particular, this sensor could potentially help validate or optimise cleaning practices for a given food manufacturing process.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Background: Protein allostery can be communicated purely through altered entropy. Results: Altered cAMP binding strength in CAP results in changes to entropy-driven allostery. Conclusion: The requirement to maintain allostery constrains evolution of the ligand-binding site in CAP. Significance: Entropy-driven processes can constrain amino acid covariation in evolution. Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

Synthesis of MIP Nanoparticles for α-Casein Detection using SPR as a Milk Allergen Sensor

Food recalls due to undeclared allergens or contamination are costly to the food manufacturing industry worldwide. As the industry strives for better manufacturing efficiencies over a diverse range of food products, there is a need for the development of new analytical techniques to improve monitoring of the presence of unintended food allergens during the food manufacturing process. In particular, the monitoring of wash samples from cleaning in place systems (CIP), used in the cleaning of food processing equipment, would allow for the effective removal of allergen containing ingredients in between food batches. Casein proteins constitute the biggest group of proteins in milk and hence are the most common milk protein allergen in food ingredients. As such, these proteins could present an ideal analyte for cleaning validation. In this work, molecularly imprinted polymer nanoparticles (nanoMIPs) with high affinity toward bovine α-casein were synthesized using a solid-phase imprinting method. The nanoMIPs were then characterized and incorporated into label free surface plasmon resonance (SPR) based sensor. The nanoMIPs demonstrated good binding affinity and selectivity toward α-casein (KD ∼ 10 × 10-9 M). This simple affinity sensor demonstrated the quantitative detection of α-casein achieving a detection limit of 127 ± 97.6 ng mL-1 (0.127 ppm) which is far superior to existing commercially available ELISA kits. Recoveries from spiked CIP wastewater samples were within the acceptable range (87-120%). The reported sensor could allow food manufacturers to adequately monitor and manage food allergen risk in food processing environments while ensuring that the food produced is safe for the consumer.