Radiation damage inflicted on macromolecular crystals during X-ray diffraction experiments remains a limiting factor for structure solution, even when samples are cooled to cryotemperatures (~100 K). ...Efforts to establish mitigation strategies are ongoing and various approaches, summarized below, have been investigated over the last 15 years, resulting in a deeper understanding of the physical and chemical factors affecting damage rates. The recent advent of X-ray free electron lasers permits "diffraction-before-destruction" by providing highly brilliant and short (a few tens of fs) X-ray pulses. New fourth generation synchrotron sources now coming on line with higher X-ray flux densities than those available from third generation synchrotrons will bring the issue of radiation damage once more to the fore for structural biologists.
Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. ...The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of ...choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D
1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X‐ray free electron lasers have facilitated investigations of structural ...dynamics in proteins with unprecedented temporal and spatial resolution. Light‐activated proteins are attractive targets for time‐resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light‐absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small‐scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time‐resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light‐activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time‐resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time‐resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.
Dynamical changes in light‐activated proteins can be dissected using the powerful techniques of time‐resolved serial femtosecond crystallography and small/wide‐angle X‐ray scattering. Light‐induced changes occur across multiple timescales, giving rise to different intermediates in the photocycle. This review highlights the use of TR‐serial femtosecond crystallography to study early events in the chromophore‐binding pocket of various proteins and discusses the use of X‐ray solution scattering techniques to probe conformational changes in proteins.
Insect carboxylesterases from the aEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid ...metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxy leste rases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcaEl in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (~10⁶ M⁻¹ s⁻¹) indicative of a natural substrate.
Radiation damage induced by X‐ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven ...in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomic B‐factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X‐ray free‐electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X‐ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field.
Significance Protein aggregation into amyloid fibers and oligomers is observed in a variety of neurodegenerative diseases. The fibers formed by the intrinsically disordered human protein tau, for ...instance, are one of the hallmarks of Alzheimer disease. In this work, we report on the dynamic behavior of tau hydration water, which we found to be more mobile in tau fibers than in nonaggregated tau. This increase in mobility could promote fiber formation through an increase in hydration water entropy. That hydration water is more mobile around the pathological form of tau corroborates that methodologies sensitive to the diffusion of water, such as diffusion magnetic resonance imaging, could be used to diagnose Alzheimer patients in an early stage of the disease.
The paired helical filaments (PHF) formed by the intrinsically disordered human protein tau are one of the pathological hallmarks of Alzheimer disease. PHF are fibers of amyloid nature that are composed of a rigid core and an unstructured fuzzy coat. The mechanisms of fiber formation, in particular the role that hydration water might play, remain poorly understood. We combined protein deuteration, neutron scattering, and all-atom molecular dynamics simulations to study the dynamics of hydration water at the surface of fibers formed by the full-length human protein htau40. In comparison with monomeric tau, hydration water on the surface of tau fibers is more mobile, as evidenced by an increased fraction of translationally diffusing water molecules, a higher diffusion coefficient, and increased mean-squared displacements in neutron scattering experiments. Fibers formed by the hexapeptide ³⁰⁶VQIVYK ³¹¹ were taken as a model for the tau fiber core and studied by molecular dynamics simulations, revealing that hydration water dynamics around the core domain is significantly reduced after fiber formation. Thus, an increase in water dynamics around the fuzzy coat is proposed to be at the origin of the experimentally observed increase in hydration water dynamics around the entire tau fiber. The observed increase in hydration water dynamics is suggested to promote fiber formation through entropic effects. Detection of the enhanced hydration water mobility around tau fibers is conjectured to potentially contribute to the early diagnosis of Alzheimer patients by diffusion MRI.
The coupling between protein dynamics and hydration-water dynamics was assessed by perdeuteration, temperature-dependent neutron scattering, and molecular dynamics simulations. Mean square ...displacements of water and protein motions both show a broad transition at 220 K and are thus coupled. In particular, the protein dynamical transition appears to be driven by the onset of hydration-water translational motion.
Abstract Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B 12 ) can act as a light-sensing chromophore ...heralded a new field of B 12 -photobiology. Although microbial genome analysis indicates that photoactive B 12 -binding domains form part of more complex protein architectures, regulating a range of molecular–cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B 12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B 12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B 12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B 12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Abstract
CarH is a coenzyme B
12
-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B
12
Co-C bond culminates in CarH tetramer dissociation ...to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B
12
-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B
12
-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B
12
photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
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