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1.
IUCrJ ; 11(Pt 2): 237-248, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38446456

RESUMO

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.


Assuntos
Arabidopsis , Microfluídica , Cristalografia , Cognição , Convecção
2.
Proc Natl Acad Sci U S A ; 121(11): e2312596121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38437555

RESUMO

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.


Assuntos
5-Metilcitosina , DNA , Cristalização , Catálise , Cristalografia
3.
Protein Sci ; 33(4): e4957, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501509

RESUMO

The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time-resolved studies, both at x-ray free-electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Cristalografia , Temperatura , NAD , Antineoplásicos/química , Flavinas , Cristalografia por Raios X , NAD(P)H Desidrogenase (Quinona)
4.
Biophys J ; 123(5): 622-637, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38327055

RESUMO

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.


Assuntos
Hidroximetilglutaril-CoA Redutases , NAD , Hidroximetilglutaril-CoA Redutases/química , Hidroximetilglutaril-CoA Redutases/metabolismo , NAD/metabolismo , Cristalografia , Ácido Mevalônico/metabolismo , Concentração de Íons de Hidrogênio , Cinética
5.
Nature ; 626(8000): 905-911, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38355794

RESUMO

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.


Assuntos
Artefatos , Lasers , Mioglobina , Cristalografia/instrumentação , Cristalografia/métodos , Elétrons , Mioglobina/química , Mioglobina/metabolismo , Mioglobina/efeitos da radiação , Fótons , Conformação Proteica/efeitos da radiação , Teoria Quântica , Raios X
6.
IUCrJ ; 11(Pt 2): 190-201, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38327201

RESUMO

Serial crystallography (SX) has become an established technique for protein structure determination, especially when dealing with small or radiation-sensitive crystals and investigating fast or irreversible protein dynamics. The advent of newly developed multi-megapixel X-ray area detectors, capable of capturing over 1000 images per second, has brought about substantial benefits. However, this advancement also entails a notable increase in the volume of collected data. Today, up to 2 PB of data per experiment could be easily obtained under efficient operating conditions. The combined costs associated with storing data from multiple experiments provide a compelling incentive to develop strategies that effectively reduce the amount of data stored on disk while maintaining the quality of scientific outcomes. Lossless data-compression methods are designed to preserve the information content of the data but often struggle to achieve a high compression ratio when applied to experimental data that contain noise. Conversely, lossy compression methods offer the potential to greatly reduce the data volume. Nonetheless, it is vital to thoroughly assess the impact of data quality and scientific outcomes when employing lossy compression, as it inherently involves discarding information. The evaluation of lossy compression effects on data requires proper data quality metrics. In our research, we assess various approaches for both lossless and lossy compression techniques applied to SX data, and equally importantly, we describe metrics suitable for evaluating SX data quality.


Assuntos
Algoritmos , Compressão de Dados , Cristalografia , Compressão de Dados/métodos , Tomografia Computadorizada por Raios X
7.
PLoS Comput Biol ; 20(2): e1011519, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38324587

RESUMO

ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) simulations combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPP's RT loop to engage with p53's C-terminal linker-a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues qualitatively overlap on ASPP proteins, and the ensemble-average binding free energies better match experimental KD values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallography-determined one. Lastly, we showed that the ensemble binding complexes are sensitive to p53's intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).


Assuntos
Proteínas Reguladoras de Apoptose , Proteína Supressora de Tumor p53 , Proteínas Reguladoras de Apoptose/química , Proteína Supressora de Tumor p53/química , Cristalografia , Ligação Proteica , Apoptose
8.
Acta Crystallogr A Found Adv ; 80(Pt 1): 1-17, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38189437

RESUMO

Deep learning techniques can recognize complex patterns in noisy, multidimensional data. In recent years, researchers have started to explore the potential of deep learning in the field of structural biology, including protein crystallography. This field has some significant challenges, in particular producing high-quality and well ordered protein crystals. Additionally, collecting diffraction data with high completeness and quality, and determining and refining protein structures can be problematic. Protein crystallographic data are often high-dimensional, noisy and incomplete. Deep learning algorithms can extract relevant features from these data and learn to recognize patterns, which can improve the success rate of crystallization and the quality of crystal structures. This paper reviews progress in this field.


Assuntos
Aprendizado Profundo , Cristalografia , Algoritmos , Comportamento Compulsivo , Cristalização
9.
Nature ; 626(7999): 670-677, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38297122

RESUMO

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.


Assuntos
Oxigênio , Complexo de Proteína do Fotossistema II , Biocatálise/efeitos da radiação , Cálcio/metabolismo , Cristalografia , Transporte de Elétrons/efeitos da radiação , Elétrons , Manganês/metabolismo , Oxirredução/efeitos da radiação , Oxigênio/química , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/efeitos da radiação , Prótons , Fatores de Tempo , Tirosina/metabolismo , Água/química , Água/metabolismo
10.
Eur J Med Chem ; 266: 116126, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38232464

RESUMO

Due to their structural diversities and prevalent biological activities, natural products (NPs) are momentous resources for drug discovery. Although NPs have a wide range of biological activities, many exhibit structural complexity that leads to synthetic difficulties, which combines with inefficient biological activity, toxicity, and unfavorable pharmacokinetic characteristics and ultimately imparts poor safety and efficacy outcomes. Progress in crystallization and computational techniques allow crystallography to have a seasonable influences on drug discovery. By co-crystallizing with proteins, therapeutic targets of NPs in specific diseases can be identified. By analyzing the co-crystal information, the structure-activity relationships (SARs) of NPs targeting specific proteins can be grasped. Under the guidance of co-crystal information, directional structural modification and simplification are powerful strategies for overcoming limitations of NPs, improving the success rate of NP-based drug discovery, and obtaining NP-based drugs with high selectivity, low toxicity and favorable pharmacokinetic characteristics. Here, we review the co-crystal information of a selection of NPs, focusing on the SARs of NPs reflected by co-crystal information and the modification and simplification strategies of NPs, and discuss how to apply co-crystal information in the optimization of NP-based lead compound.


Assuntos
Produtos Biológicos , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Descoberta de Drogas/métodos , Relação Estrutura-Atividade , Cristalografia
11.
Acta Crystallogr D Struct Biol ; 80(Pt 2): 60-79, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38265875

RESUMO

Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump-probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.


Assuntos
Proteínas , Síncrotrons , Cristalografia , Cristalografia por Raios X , Raios X , Proteínas/química , Lasers
12.
Urolithiasis ; 52(1): 28, 2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38244096

RESUMO

The relationship between urinary tract infection caused by urease-producing bacteria and lithiasis due to struvite stones is well established in the literature. However, there is limited knowledge on whether non-urease producing bacteria can also promote crystallization. In our study, we analyzed the association between urinary lithiasis, other than struvite by crystallography and non-ureolytic bacteria, in 153 patients who underwent surgery for urinary stone. The collected samples were sent for crystallographic analysis and culture. Additionally, preoperatory urine culture was collected for combined evaluation with the previous data. Percutaneous nephrolithotomy was the most commonly performed approach (45.8%). Struvite stones were more frequently identified in women (90.3%). Among stones with positive cultures, except struvite, 45.5% were composed of calcium oxalate monohydrate. The difference between urine culture and stone culture was different in 24.8% of the cases. Among stones with positive cultures that did not contain struvite, 86.4% showed non-urease bacteria in their cultures and 47.1% of struvite stones also did not have urease-producing bacteria in their cultures (p < 0.021). Our findings suggest that there is an association between non-ureolytic bacteria and stones that are not composed of struvite.


Assuntos
Cálculos Urinários , Urolitíase , Humanos , Feminino , Estruvita , Cristalografia , Urease , Urolitíase/complicações , Cálculos Urinários/urina , Bactérias
14.
Dent Mater ; 40(2): 267-275, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37989699

RESUMO

OBJECTIVES: The aim of this work is to test experimental cements, doped with a silicate based bioactive nanoparticle (NanoBiosilicate). Methods, we synthesized a glass nanoparticle by Sol-Gel Stöber method, used to be incorporated in a dental material for endodontic uses. MATERIALS AND METHODS: We assess the mineralizing properties and biocompatibility. Besides the crystallography characterization of the resultant new crystals. Results, After analysis, and comparison with commercial materials, the material tested was similar in mechanical properties required by ISO, The ion release was effective after 2 hr. of setting and the novel material was cell compatible accepted by ISO. RESULTS: We found new formed Calcium Phosphate peaks in the spectroscopic analysis (FTIR), remarkably the crystals formed were comparable to hydroxyapatite when analyzed with a Selected Area Electron Diffractometer, with rings of 2.84 Å for 002, and the 2.77 Å is also visible for 210. The 6.83 Å and 6.88 Å, for respective 222 and 004. The incorporation of Chlorhexidine was not detrimental for this property, Significance, the features mentioned represented a progress in biomineralization field that was associated to an improved mineral structure formation with increased crystallographic similarity to natural hydroxyapatite. When chlorhexidine was added a favorable biomodification of the remaining collagen in dentinal walls and antimicrobial activity potential were also observed.


Assuntos
Compostos de Cálcio , Durapatita , Compostos de Cálcio/química , Clorexidina/farmacologia , Cristalografia , Biomineralização , Teste de Materiais , Fosfatos de Cálcio/química , Silicatos/química , Colágeno
15.
J Prosthet Dent ; 131(1): 164.e1-164.e11, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37945513

RESUMO

STATEMENT OF PROBLEM: High translucency zirconia (HTZ) has gained popularity as an esthetic computer-aided design and computer-aided manufacturing (CAD-CAM) material for monolithic restorations. A detailed comparison between different common surface and heat treatments with a non-treated HTZ control to explain the behavior of the material under stress is lacking. PURPOSE: The purpose of this in vitro study was to evaluate the effect of different surface and heat treatments on the surface roughness parameters (SRPs), topography, crystallography, and phase composition of HTZ used for monolithic restorations. MATERIAL AND METHODS: Ninety Ø11.9×1.18-mm HTZ disks (Prettau Anterior) were milled, sintered, and distributed into 9 groups (n=10); 8 experimental (coarse diamond grinding GC, fine diamond grinding GF, fine diamond grinding and 3-step polishing kit GF+P1, fine diamond grinding and 3-step polishing kit and diamond paste GF+P1+DP, fine diamond grinding and 2-step polishing kit GF+P2, fine diamond grinding and GF+Gl, fine diamond grinding and 3-step polishing and glazing GF+P1+Gl, airborne-particle abrasion with 50-µm alumina), and a control group (C, as-sintered). SRPs (AveSa, AveSv, AveSz) and 3-dimensional (3D) images were obtained using a noncontact 3D-optic-profilometer. The crystal structure was determined with scanning electron microscopy. Phase composition was analyzed by X-ray diffraction (XRD). Surface roughness parameters data were statistically analyzed by 1-way ANOVA and the Tukey HSD test (α=.05). RESULTS: The applied surface and heat treatment resulted in significantly different SRP mean values (P<.001) with different topographies. GC had the highest AveSa, AveSv, and AveSz mean values (0.95, 8.8, 17.4 µm, respectively) with significant microcracks. GF had significantly lower SRP with finer microcracks. GF+P1 had a significantly smoother surface, but GF+P2 resulted in SRP comparable with the GF group. GF+P1+DP had the smoothest homogenous surface (mean Sa: 0.08 µm). GF+P1 and GF+GL were equally effective, while GF+P1+GL was not superior. Airborne-particle abrasion produced a low Sa mean value (0.11 µm) with relatively high Sv and Sz mean values (5.9, 9.2 µm, respectively) and microcracks. A monoclinic phase was detected in all groups. All experimental groups had broadened XRD-peaks with lower intensity, suggesting the presence of the rhombohedral phase. CONCLUSIONS: The different surface and heat treatments altered the HTZ crystals and their surface roughness with distinct topographies. Cubic crystal changes take place under stress as shown by the scanning electron microscope and the XRD diffraction pattern and may transform to the rhombohedral phase.


Assuntos
Polimento Dentário , Temperatura Alta , Teste de Materiais , Cristalografia , Polimento Dentário/métodos , Propriedades de Superfície , Estética Dentária , Zircônio/química , Microscopia Eletrônica de Varredura , Diamante/química , Cerâmica/química
16.
Nat Methods ; 21(1): 110-116, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38036854

RESUMO

Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.


Assuntos
Inteligência Artificial , Processos Mentais , Cristalografia , Conformação Proteica
18.
Curr Opin Struct Biol ; 84: 102741, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38086321

RESUMO

Microcrystal electron diffraction, commonly referred to as MicroED, has become a powerful tool for high-resolution structure determination. The method makes use of cryogenic transmission electron microscopes to collect electron diffraction data from crystals that are several orders of magnitude smaller than those used by other conventional diffraction techniques. MicroED has been used on a variety of samples including soluble proteins, membrane proteins, small organic molecules, and materials. Here we will review the MicroED method and highlight recent advancements to the methodology, as well as describe applications of MicroED within the fields of structural biology and chemical crystallography.


Assuntos
Elétrons , Proteínas de Membrana , Microscopia Crioeletrônica/métodos , Cristalografia/métodos
19.
Acta Crystallogr D Struct Biol ; 80(Pt 1): 16-25, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38088897

RESUMO

The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm-2, providing experimental laser and delay parameters for a successful TR-MX experiment.


Assuntos
Proteínas , Síncrotrons , Análise Espectral , Proteínas/química , Cristalografia , Luz
20.
Sci Adv ; 9(49): eadh4179, 2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38064560

RESUMO

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.


Assuntos
Monóxido de Carbono , Complexo IV da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Domínio Catalítico , Monóxido de Carbono/química , Cristalografia , Oxirredução , Oxigênio/metabolismo
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