Biology subjects

Pudney, C. R.

Publications and source records attributed to Pudney, C. R..

10 recordsLinked to original sources

bioRxiv2025

Environmental dipolar relaxation during excited state proton transfer in Green Fluorescent Protein

Engineered variants of Green Fluorescent Protein (GFP) are in widespread use as genetically-encoded labels for biological imaging. Excitation of its neutral phenolic ground state chromophore populates an emissive anionic phenolate state via excited state proton transfer (ESPT) to E222 along a molecular wire. Computational studies indicate that ESPT fits to an electronically adiabatic rate expression. Although reactions that operate close to the adiabatic limit can be sensitive to environmental dipolar relaxation, little is known about the role of such relaxation during ESPT in GFP. Here, we present the first experimental evidence that dipolar relaxation of the protein matrix occurs during ESPT in response to the change in electron density distribution in the excited state GFP chromophore and is a key determinant of the reaction pathway. Using fluorescence spectroscopy, we excited along the red edge of the neutral phenolic ground state absorption band of several GFP variants with differing chromophore environments. Instead of resulting in a significant red shift of the centre of spectral mass (CSM) of the emission spectra common for biological chromophores such as tryptophan, the CSM of each GFP variant remains almost unchanged as a function of excitation wavelength. This is consistent with each sub-population that is selectively excited along the red edge reaching the same, fully relaxed state before emission of a photon. The kinetics of environmental dipolar relaxation are therefore on the same sub-nanosecond timescale as ESPT, which provides an explanation for its adiabatic nature and could inform the rational design of novel fluorescent proteins with tailored photophysics.

biophysics↗

bioRxiv2024

Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

biochemistry↗

bioRxiv2023

Cooperative conformational transitions and the temperature dependence of enzyme catalysis

Many enzymes display non-Arrhenius behaviour with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behaviour and recently the role of the activation heat capacity [Formula] has been widely discussed. If enzyme-catalysed reactions occur with appreciable negative values of [Formula] (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalysed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterise the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallised the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 [A]). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalises disparate behaviour seen for MalL and a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterise the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilisation that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity, and other aspects of enzyme catalysis.

biochemistry↗

bioRxiv2022

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here we show glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its activity and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRPs thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its non-glycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data shows that glycosylation does not just have a passive effect on HRP stability but can exert long range effects that mediate the native enzymes activity and stability through changes in inherent dynamics.

biochemistry↗

bioRxiv2021

A thermodynamic model for interpreting tryptophan excitation-energy-dependent fluorescence spectra provides insight into protein conformational sampling and stability

It is now over thirty years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. Whilst there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the protein REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides to multi-tryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibody in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating bimolecular simulation with experimental data to yield novel insights.

biophysics↗

bioRxiv2021

Chemical mapping exposes the importance of active site interactions in governing the temperature dependence of enzyme turnover

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, {Delta}CP{ddagger}, can reveal links between the protein free energy landscape, global protein dynamics and enzyme turnover, suggesting that subtle changes molecular interactions at the active site can affect long range protein dynamics and link to enzyme temperature activity. Here we use a model promiscuous enzyme (Glucose dehydrogenase from Sulfolobus Solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, REES spectroscopy and computational simulation we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in {Delta}CP{ddagger} and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

biochemistry↗

bioRxiv2021

Reliable in silico ranking of engineered therapeutic TCR binding affinities using MMPBSA and MMGBSA calculations.

Accurate and efficient in silico ranking of protein-protein binding affinities is useful for protein design with applications in biological therapeutics. One popular approach to rank binding affinities is to apply the molecular mechanics Poisson Boltzmann/generalized Born surface area (MMPB/GBSA) method to molecular dynamics trajectories. Here, we identify protocols that enable the reliable evaluation of T-cell receptor (TCR) variants binding to their target, peptide-human leukocyte antigens (pHLAs). We suggest different protocols for variant sets with few ([≤]4) or many mutations, with entropy corrections important for the latter. We demonstrate how potential outliers could be identified in advance and that just 5-10 replicas of short (4 ns) MD simulations may be sufficient for reproducible and accurate ranking of TCR variants. The protocols developed here can be applied towards in silico screening during the optimization of therapeutic TCRs, potentially reducing both the cost and time taken for biologic development.

biophysics↗

bioRxiv2021

Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity

Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating a de novo peroxidases activity. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.

biochemistry↗

bioRxiv2020

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a fingerprint of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (g-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

biochemistry↗

bioRxiv2020

How to make a dial not a switch: Control of histidine kinase conformation by an ABC-transporter facilitates need-based activation of antibiotic resistance

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

microbiology↗