Challenges and solutions for the analysis of<i>in situ</i>,<i>in crystallo</i>micro-spectrophotometric data

Combining macromolecular crystallography within crystallomicro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and theSLS-APEin situ,in crystallooptical spectroscopy data-analysis toolbox is demonstrated.</jats:p

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when appliedin situat macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochromec′ fromAlcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.</jats:p

A subtle structural change in the distal haem pocket has a remarkable effect on tuning hydrogen peroxide reactivity in dye decolourising peroxidases from Streptomyces lividans

Dye decolourising peroxidases (DyPs) are oxidative haem containing enzymes that can oxidise organic substrates by first reacting with hydrogen peroxide. Herein, we have focused on two DyP homologs, DtpAa and DtpA, from the soil-dwelling bacterium Streptomyces lividans. By using X-ray crystallography, stopped-flow kinetics, deuterium kinetic isotope studies and EPR spectroscopy, we show that both DyPs react with peroxide to form Compound I (a FeIV=O species and a porphyrin π-cation radical), via a common mechanism, but the reactivity and rate limits that define the mechanism are markedly different between the two homologs (DtpA forms Compound I rapidly, no kinetic isotope effect; DtpAa 100-fold slower Compound I formation and a distinct kinetic isotope effect). By determining the validated ferric X-ray structure of DtpAa and comparing it with the ferric DtpA structure, we attribute the kinetic differences to a subtle structural repositioning of the distal haem pocket Asp side chain. Through site-directed mutagenesis we show the acid-base catalyst responsible for proton-transfer to form Compound I comprises a combination of a water molecule and the distal Asp. Compound I formation in the wild-type enzymes as well as their distal Asp variants is pH dependent, sharing a common ionisation equilibrium with an apparent pKa of ~ 4.5-5.0. We attribute this pKa to the deprotonation/protonation of the haem bound H₂O₂. Our studies therefore reveal a mechanism for Compound I formation in which the rate limit may be shifted from peroxide binding to proton-transfer controlled by the distal Asp position and the associated hydrogen-bonded water molecules

Serial femtosecond zero dose crystallography captures a water‐free distal heme site in a dye‐decolourising peroxidase to reveal a catalytic role for an arginine in FeIV=O formation

Obtaining structures of intact redox states of metal centres derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolourising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues, aspartate and arginine, in the heterolysis of peroxide to form the catalytic intermediate compound I (Fe IV =O and a porphyrin cation radical). Using serial femtosecond X‐ray (SFX) crystallography, we have determined the pristine structures of the Fe III and Fe IV =O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site, which together with the presence of an asparagine, infer the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis

Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector

Observations of the dose-rate effect in continuous X-ray diffraction data acquisition at room temperature are presented

Ultrafast structural changes direct the first molecular events of vision

視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation

X-RAY INDUCED PHOTODAMAGE IN PROTEINS: A RAMAN-ASSISTED BIOCRYSTALLOGRAPHIC STUDY

Currently, few sites provide the possibility to perform simultaneous Raman/X-ray diffraction of single macromolecular crystals. In these recent Raman-assisted crystallography applications, Raman microscopy has been representing a fine servant of a dominant X-ray Crystallography, and its highest scope was the reinforcement of structural data [1, 2]. The strategy of our work is to overturn this paradigm, focusing Crystallography-assisted Raman on the rich spectroscopic data that are well transferable to many bio-analytical applications. A Synchrotron with available Raman microscope, SLS [3], has been used to collect high-resolution crystallographic data on unusual states of model biomolecules. X-ray induced radiation damage is a frequent phenomenon, especially when using third generation synchrotrons. Raman microscopy prior and after X-ray data collection can be a valuable tool to detect artifacts derived from radiation damage. A case of study is presented: ultra high resolution crystal structure (0.8 Å) of ribonuclease A at different X- ray doses. Herein, we provide ultra high resolution data (0.8 Å resolution) at six different doses (from 0,4 up to 24 MGy), with a clear disappearance of Raman bands due to photodamage. We observe, studying RNase A crystals, novel Raman bands (photoproducts) appearing upon X-ray dose increases. Ultra-high resolutions provide electron density maps of such compounds derived from degradation of methionine, aspartic acids and tyrosine side chains. Some of these appearing bands have been identified, whereas other Raman markers are still under investigation, combining Raman data with ultra-high resolution crystallography. Ultra-high resolutions have provided electron density maps of compounds derived from degradation of methionine and tyrosines. Some of these appearing bands have been identified, e.g. methyl radicals at 1356 cm-1 . Other Raman markers are still under investigation (987 cm-1)

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source

The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years.ISSN:0909-0495ISSN:1600-577