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With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid "photochemical fingerprinting"approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device.

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-HRP’s 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’ enzyme’s activity and stability through changes in inherent dynamics.

Here, we report a label-free gold nanoparticle-based single-molecule optical platform to study the immobilization, activity, and thermodynamics of single enzymes. The sensor uses plasmonic gold nanoparticles coupled to optical whispering gallery modes (WGMs) to probe enzyme conformational dynamics during turnover at a microsecond time resolution. Using a glucosidase enzyme as the model system, we explore the temperature dependence of the enzyme turnover at the single-molecule (SM) level. A recent physical model for understanding enzyme temperature dependencies (macromolecular rate theory; MMRT) has emerged as a powerful tool to study the relationship between enzyme turnover and thermodynamics. Using WGMs, SM enzyme measurements enable us to accurately track turnover as a function of conformational changes and therefore to quantitatively probe the key feature of the MMRT model, the activation heat capacity, at the ultimate level of SM. Our data shows that WGMs are extraordinarily sensitive to protein conformational change and can discern both multiple steps with turnover as well as microscopic conformational substates within those steps. The temperature dependence studies show that the MMRT model can be applied to a range of steps within turnover at the SM scale that is associated with conformational change. Our study validates the notion that MMRT captures differences in dynamics between states. The WGM sensors provide a platform for the quantitative analysis of SM activation heat capacity, applying MMRT to the label-free sensing of microsecond substates of active enzymes.

BACKGROUND AND AIMS: The United Kingdom (UK) Psychoactive Substances Act (PSA), implemented on the 26 th May 2016, made the production, supply and sale of all non-exempted psychoactive substances illegal. The aim of this study was to measure trends in hospital presentations for severe toxicity following analytically confirmed synthetic cannabinoid receptor agonist (SCRA) exposure before and after implementation of the PSA. DESIGN: Observational study.SETTING: Thirty-four hospitals across the UK participating in the Identification of Novel Psychoactive Substances (IONA) study.PARTICIPANTS: A total of 627 (79.9% male) consenting individuals who presented to participating hospitals between July 2015 and December 2019 with severe acute toxicity and suspected novel psychoactive substances exposure.MEASUREMENTS: Toxicological analyses of patient samples were conducted using liquid-chromatography tandem mass-spectrometry. Time-series analysis was conducted on the monthly number of patients with and without analytically confirmed SCRA exposure using Poisson segmented regression.FINDINGS: SCRAs were detected in 35.7% (n = 224) of patients. After adjusting for seasonality and the number of active sites, models showed no clear evidence of an upward or downward trend in the number of SCRA exposure cases in the period before (incidence rate ratio [IRR], 1.12; 95% CI, 0.99-1.26; P = 0.068) or after (IRR, 0.97; 95% CI, 0.94-1.01; P = 0.202) the implementation of the PSA. There was also no clear evidence of an upward or downward trend in non-SCRA exposure cases before (IRR, 1.12; 95% CI, 0.98-1.27; P = 0.105) or after (IRR, 1.01; 95% CI, 0.98-1.04; P = 0.478) implementation of the PSA.CONCLUSIONS: There is no clear evidence of an upward or downward trend in the number of patients presenting to UK hospitals with severe acute toxicity following analytically confirmed synthetic cannabinoid receptor agonist exposure since the implementation of the Psychoactive Substances Act.

Experiments and biomolecular simulations are revealing new and unexpected details of how enzymes are adapted to specific temperatures. These findings are elucidating enzyme evolutionary trajectories and offer great promise for design and engineering of natural and artificial enzymes. They also have implications for understanding responses of larger scale biological temperature dependence, relevant for understanding the effects of climate change on ecosystems. We review recent work on the temperature dependence of enzyme-catalysed reaction rates and the implications for enzyme evolution. Evidence from kinetic isotope effects, temperature dependent reaction rates, molecular dynamics simulations and thermodynamics provides new insights into enzyme thermoadaptation and evolution.

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.

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 (μg–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.

Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug‐target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αβ T‐cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T‐cell compartment. We have applied high‐pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide–HLA complexes. We find that different peptide sequences affect peptide–HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide‐binding interface, in a manner that could influence T‐cell antigen discrimination., TCRs recognise peptide–HLA complexes mediating immune reactivity against pathogens and neoplasms. We investigated the drivers that govern molecular flexibility of different peptide–HLAs. We found that HLA motions were mediated by small alterations in the bound peptide, suggesting that allosteric mechanisms mediate HLA flexibility (‘the tail wags the dog’). Our study sheds new light on the molecular mechanisms guiding protein dynamics in peptide–HLA, the major target for the TCR.

Accurate and efficient

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, ΔCP‡, 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 ΔCP‡ 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.

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 activity in the de novo peroxidase C45. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending toward a more globally rigid structure. This is correlated with increases in 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 states. 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 states. 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.

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Using computational design, we converted metalloporphyrin specificity of cytochrome b562 from iron to fluorogenic zinc. The new variant had a near total preference for zinc representing a switch in specificity, which greatly enhanced the negligible aqueous fluorescence of free ZnPP in vitro and in vivo.

Synthetic cannabinoid receptor agonists (SCRAs), termed “Spice” or “K2”, are molecules that emulate the effects of the active ingredient of marijuana, and they have gained enormous popularity over the past decade. SCRAs are Schedule 1 drugs that are highly prevalent in the U.K. prison system and among homeless populations. SCRAs are highly potent and addictive. With no way to determine the dose/amount at the point-of care, they pose severe health risks to users, including psychosis, stroke, epileptic seizures, and they can kill. SCRAs are chemically diverse, with over a hundred compounds used as recreational drugs. The chemical diversity of SCRA structures presents a challenge in developing detection modalities. Typically, GC-MS is used for chemical identification; however, this cannot be in place in most settings where detection is critical, e.g., in hospital Emergency Departments, in custody suites/prisons, or among homeless communities. Ideally, real time, point-of-care identification of SCRAs is desirable to direct the care pathway of overdoses and provide information for informed consent. Herein, we show that fluorescence spectral fingerprinting can be used to identify the likely presence of SCRAs, as well as provide more specific information on structural class and concentration (∼1 μg mL–1). We demonstrate that that fluorescence spectral fingerprints, combined with numerical modeling, can detect both parent and combusted material, and such fingerprinting is also practical for detecting them in oral fluids. Our proof-of-concept study suggests that, with development, the approach could be useful in a range of capacities, notably in harm reduction for users of Spice/K2.

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 peroxidase’s 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.

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1, which has an apparent melting temperature of 26{degrees}C. EstN7 is a dimer with a classical /{beta} hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. However, dynamics do not appear to play a major role in cold adaption. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests there is little difference in dynamics with the catalytically important N-terminal cap comprising the main dynamic element. Molecular dynamics, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. The conformation of the cap region is significantly different to EstN7s closest relatives, forming a bridge-like structure with reduced helical content providing more than one access tunnel through to the active site. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7s cold adaptation rather than changes in dynamics.

Synthetic cannabinoids are a major class of NPS with a high impact on analytical, forensic and toxicological fields. The main route of administration of synthetic cannabinoids is through inhalation, delivered by smoking a conventional or electronic cigarette, pyrolysis possibly altering the nature of these compounds and resulting in unknown combustion products with unknown biological activity/toxicology. In this study, a twin-trap smoking inhalation model that simulates human inhalation has been developed. The smoking simulator allows the efficient trapping of the combustion products. Analysis using UHPLC-TOF-ESI-MS and GC-MS allowed the detection of all six synthetic cannabinoids tested in the smoking simulator. A novel combustion product of MDMB-CHMICA was identified, a dihydro-1H-pyridoindole-dione with its structure and mechanism of formation proposed. This study of SC combustion products provides markers which may be useful for drug analysts and toxicologists in examining samples obtained from suspected SC users/abusers.

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

Understanding how enzyme catalysis varies with temperature is key to understanding catalysis itself and, ultimately, how to tune temperature optima. Temperature dependence studies inform on the change in heat capacity during the reaction, ΔCP‡, and we have recently demonstrated that this can expose links between the protein free energy landscape and enzyme turnover. By quantifying ΔCP‡, we capture information on the changes to the distribution of vibrational frequencies during enzyme turnover. The primary experimental tool to probe the role of vibrational modes in a chemical/biological process is isotope effect measurements, since isotopic substitution primarily affects the frequency of vibrational modes at/local to the position of isotopic substitution. We have monitored the temperature dependence of a range of isotope effects on the turnover of a hyper-thermophilic glucose dehydrogenase. We find a progressive effect on the magnitude of ΔCP‡ with increasing isotopic substitution of d-glucose. Our experimental findings, combined with molecular dynamics simulations and quantum mechanical calculations, demonstrate that ΔCP‡ is sensitive to isotopic substitution. The magnitude of the change in ΔCP‡ due to substrate isotopic substitution indicates that small changes in substrate vibrational modes are “translated” into relatively large changes in the (distribution and/or magnitude of) enzyme vibrational modes along the reaction. Therefore, the data suggest that relatively small substrate isotopic changes are causing a significant change in the temperature dependence of enzymatic rates.

Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach.

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Biology uses a narrow set of metal centres comprising mainly of iron and magnesium. Here, we convert metalloporphyrin specificity of cytochrome b562 from iron (haem) to fluorogenic zinc protoporphyrin IX (ZnPP). Through a computationally guided iterative design process, a variant with a near total preference for ZnPP was generated representing a switch in specificity. The new variant greatly enhanced (≥60 fold) the negligible aqueous fluorescence of free ZnPP in vitro and in vivo.Abstract Figure

There is an increasing realization that structure-based drug design may show improved success rates by understanding the ensemble of conformations and sub-states accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson’s disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear and routes to drugging this target would be valuable and relevant. Evidence of a radical in either the transition state or resting state of MAO-B is present throughout the literature, and is suggested to be a flavin semiquinone, a tyrosyl radical or both. Here we see evidence of a resting state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. Based on enzyme kinetic studies, enzyme variants and molecular dynamics simulations we find evidence for the crucial importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to effects on the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity, protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these drug targets.

There is now crucial medical importance placed on understanding the role of early stage, subvisible protein aggregation, particularly in neurodegenerative disease. While there are strategies for detecting such aggregates in vitro, there is no approach at present that can detect these toxic species associated with cells and specific subcellular compartments. We have exploited excitation-energy-dependent fluorescence edge-shift of recombinant protein labeled with a molecular beacon, to provide a sensitive read out for the presence of subvisible protein aggregates. To demonstrate the potential utility of the approach, we examine the major peptide associated with the initiation of Alzheimer's disease, amyloid β-protein (Aβ) at a patho-physiologically relevant concentration in mouse cortical neurons. Using our approach, we find preliminary evidence that subvisible Aβ aggregates are detected at specific subcellular regions and that neurons drive the formation of specific Aβ aggregate conformations. These findings therefore demonstrate the potential of a novel fluorescence-based approach for detecting and imaging protein aggregates in a cellular context, which can be used to sensitively probe the association of early stage toxic protein aggregates within subcellular compartments.

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalysed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalysed reactions occur with significant values of ∆Cp(‡) that are in general negative. That is, the heat capacity (∆Cp) for the enzyme-substrate complex is generally larger than ∆Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis (Pauling), a negative value for ∆Cp(‡) is the result of the enzyme binding weakly to the substrate and very tightly to the transition state. This observation of negative ∆Cp(‡) has important implications for the temperature dependence of enzyme-catalysed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalise the behaviour of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes and enzymes drive much of metabolism. Therefore we also expect to see characteristics of MMRT at the level of cells, whole organisms and even ecosystems.

We report the synthesis of new isoalloxazines using a microwave-assisted approach to make N-substituted-2-nitroanilines followed by one-pot reduction and condensation via Hemmerich’s method. The influence of substituents on positions 7, 8, and 10 of flavin core on the optical properties is investigated. The aliphatic functionalities on N10 give rise to quantum yields of 0.7, while aromatic side-chains quench fluorescence. Relaxed geometries (DFT) of chiral and achiral derivatives have been used for TD-DFT calculations, which yielded good agreement with the experimental UV and CD data.

The NF-κB essential modulator (NEMO) is the master regulator of NF-κB signaling, controlling the immune and nervous systems. NEMO affects the activity of IκB kinase-β (IKKβ), which relieves the inhibition of the NF-κB transcriptional regulation machinery. Despite major effort, there is only a very sparse, phenomenological understanding of how NEMO regulates IKKβ and shows specificity in its large range of molecular interactions. We explore the key molecular interactions of NEMO using a molecular biophysics approach, incorporating rapid-mixing stopped-flow, high-pressure, and CD spectroscopies. Our study demonstrates that NEMO has a significant degree of native structural disorder and that molecular flexibility and ligand-induced conformational change are at the heart of the molecular interactions of NEMO. We found that long chain length, unanchored, linear polyubiquitin drives NEMO activity, enhancing the affinity of NEMO for IKKβ and the kinase substrate IκBα and promoting membrane association. We present evidence that unanchored polyubiquitin achieves this regulation by inducing NEMO conformational change by an allosteric mechanism. We combine our quantitative findings to give a detailed molecular mechanistic model for the activity of NEMO, providing insight into the molecular mechanism of NEMO activity with broad implications for the biological role of free polyubiquitin.

α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson's disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.

Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalyzed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule (SM) enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here, single pair fluorescence energy transfer (spFRET) was used to measure SM enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from nonenzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics. © 2013 American Chemical Society.

To understand complex molecular interactions it is necessary to account for molecular flexibility and the available equilibrium of conformational states. Only a small number of experimental approaches can access such information. Potentially steady-state red edge excitation shift (REES) spectroscopy can act as a qualitative metric of changes to the protein free energy landscape (FEL) and the equilibrium of conformational states. First we validate this hypothesis using a single Trp containing protein, NF-κB essential modulator (NEMO). We provide detailed evidence from chemical denaturation studies, macromolecular crowding studies and the first report of the pressure-dependence of the REES effect. Combined these data demonstrate that the REES effect can report on the 'ruggedness' of the FEL and we present a phenomenological model, based on realistic physical interpretations, for fitting steady-state REES data to allow quantification of this aspect of the REES effect. We test the conceptual framework we have developed by correlating findings from NEMO ligand binding studies with the REES data in a range of NEMO-ligand binary complexes. Our findings shed light on the nature of the interaction between NEMO and poly-ubiquitin, suggesting that NEMO is differentially regulated by poly-ubiqutin chain length and that this regulation occurs via a modulation of the available equilibrium of conformational states, rather than gross structural change. This study therefore demonstrates the potential of REES as a powerful tool for tackling contemporary issues in structural biology and biophysics and elucidates novel information on the structure-function relationship of NEMO and key interaction partners. This article is protected by copyright. All rights reserved.

The formation and chemistry of flavin–indole charge transfer (CT) complexes has been studied using a model cationic flavin. The ability to form a CT complex is sensitive to indole structure as gauged by spectroscopic, kinetics and crystallographic studies. Single crystals of sufficient quality of a flavin–indole CT complex, suitable for X-ray diffraction, have been grown, allowing solid-state structural analysis. When CT complex formation is conducted in d4-methanol, an efficient and synthetically useful C-3 indole deuteration is observed.

There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic/NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.

The role of dynamical effects in enzyme catalysis is both complex and widely debated. Understanding how dynamics can influence the barrier to an enzyme catalyzed reaction requires the development of new methodologies and tools. In particular compressive dynamics-the focus of this study-may decrease both the height and width of a reaction barrier. By making targeted mutations in the active site of morphinone reductase we are able to alter the equilibrium of conformational states for the reactive complex in turn altering the donor-acceptor (D-A) distance for H-transfer. The sub-A changes which we induce are monitored using novel spectroscopic and kinetic "rulers". This new way of detecting variation in D-A distance allows us to analyze trends between D-A distance and the force constant of a compressive dynamical mode. We find that as the D-A distance decreases, the force constant for a compressive mode increases. Further, we demonstrate that-contrary to current dogma-compression may not always cause the magnitude of the primary kinetic isotope effect to decrease.

We report the first study of the effects of hydrostatic pressure on α-2° KIEs for an enzyme-catalysed H-transfer reaction that occurs by ‘deep’ tunnelling. High pressure causes a significant decrease in the observed α-2° KIE on the pre-steady-state hydride transfer from NADH to FMN in the flavoprotein morphinone reductase. We have recently shown that high pressure causes a reduction in macroscopic reaction barrier width for this reaction. Using DFT vibrational analysis of a simple active site model, we posit that the decrease in α-2° KIE with pressure may arise due to a decrease in the vibrational coupling between the NADH primary (transferred) and secondary hydrogens in the ‘tunnelling ready configuration’, which more closely resembles the reactant state than the transition state. Copyright © 2010 John Wiley & Sons, Ltd.

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Elucidating the origin of substrate and coenzyme specificity has been the focus of much work relating to enzyme engineering. Many enzymes exhibit tight specificity for particular substrates despite a close structural relationship to other nonreactive compounds. This tight specificity is especially remarkable and important biologically for the coenzymes NADH and NADPH. In the present study, we examined the preference of pentaerythritol tetranitrate reductase, an 'old yellow enzyme' family member, for the coenzymes NADPH over NADH. Using structural and mutagenesis studies, we have previously established that the coenzyme nicotinamide group is the key binding determinant in old yellow enzymes [Khan H et al. (2005) FEBS J 272, 4660-4671]. We have now performed detailed transient-state studies using NAD(P)H and the nonreactive analogues 1,4,5,6-tetrahydroNAD(P)H [NAD(P)H4], leading us to uncover an additional binding step in the reductive half-reaction of pentaerythritol tetranitrate reductase. We suggest that this initial binding step may primarily reflect binding of the adenine ribophosphate portion of the coenzyme, and that the second step reflects a rearrangement of the nicotinamide. Bipartite recognition, in which the adenine ribophosphate moiety localizes the coenzyme in the active site region, enables subsequent and localized searches of configurational space by the nicotinamide moiety to form the catalytically relevant charge-transfer complex. We suggest that this localized search contributes to catalytic efficiency via the principle of 'reduction in dimensionality'.

We show, both experimentally and by kinetic modeling, that enzymatic single-turnover (pre-steady-state) H-transfer reactions can be significantly complicated by kinetic isotope fractionation. This fractionation results in the formation of more protiated than deuterated product and is a unique problem for pre-steady-state reactions. When observed rate constants are measured using rapid-mixing (e.g., stopped flow) methodologies, kinetic isotope fractionation can lead to a large underestimation of both the magnitude and temperature dependence of kinetic isotope effects (KIEs). This fractionation is related to the isotopic purity of the substrates used and highlights a major problem with experimental studies which measure KIEs with substrates that are not isotopically pure. As it is not always possible to prepare isotopically pure substrates, we describe two general methods for the correction, for known isotope impurities, of KIEs calculated from pre-steady-state measurements.

The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts. Knowledge of enzyme catalysis is predominantly descriptive, gained from traditional protein crystallography and solution studies. Our goal is to understand catalysis by developing a complete and quantitative picture of catalytic processes, incorporating dynamic aspects and the role of quantum tunnelling. Embracing ideas that we have spearheaded from our work on quantum mechanical tunnelling effects linked to protein dynamics for H-transfer reactions, we review our recent progress in mapping macroscopic kinetic descriptors to an atomistic understanding of dynamics linked to biological H-tunnelling reactions.

Using alpha-secondary kinetic isotope effects (2 degrees KIEs) in conjunction with primary (1 degrees ) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2 degrees KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2 degrees KIEs, unlike 1 degrees KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2 degrees KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1 degrees KIE. The work emphasizes the benefit of combining studies of 1 degrees and 2 degrees KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.

The measurement of kinetic isotope effects (KIEs) has proved useful in many mechanistic studies of enzyme activity, most notably in enzyme-catalyzed hydrogen-transfer reactions. Primary KIEs (1° KIE) greater than unity indicate that transfer of the hydrogen species of interest is partially or fully rate limiting, and studies of the magnitude of the temperature and pressure dependence of these KIEs can inform on the mechanism of transfer. For example, KIE measurements have proved crucial in understanding the role of quantum mechanical tunneling in enzyme systems. The measurement of secondary KIEs (2° KIEs) is also informative and can be used to infer a significant tunneling contribution and details of transition state geometry. Here the deuterium label is introduced next to that of the transferred hydrogen. Measurements of 1° and 2° KIEs are being used increasingly in studies of H-transfer in flavoprotein enzymes and this requires the preparation of high purity and stereospecific labeled isotopologues. Strategies for the synthesis of labeled substrates are dependent on the enzyme system being studied. However, the nicotinamide coenzymes are often used in studies of flavoprotein enzyme mechanisms. Here, we provide practical details for the enzymatic synthesis of high purity deuterated isotopologues of the common biological coenzymes NADH and NADPH as well as the corresponding nonreactive mimics, tetrahydroNAD(P)H. Both forms of the coenzyme have proven useful in the study of mechanisms, particularly in relation to the involvement of quantum mechanical tunneling and dynamics in enzymatic H-transfer chemistry. The focus here is on practical considerations in the synthesis of these compounds. We also provide an abbreviated description of how measurements of KIEs can inform on flavoprotein mechanisms. The aim of this contribution is not to give a detailed description of the underlying theory (which has been reviewed extensively in the literature), but to provide a basic introduction and practical considerations for nonexpert readers who wish to incorporate such measurements in studies of enzyme mechanisms.