Time course of chemical and structural events in protein crystals measured by microspectrophotometry

1992 ◽  
Vol 340 (1657) ◽  
pp. 191-207 ◽  
Keyword(s):  
Time Course ◽  
Equilibrium Distribution ◽  
Synergistic Effects ◽  
Conformational Transitions ◽  
Structural Studies ◽  
Time Resolved ◽  
The Past ◽  
Catalytic Intermediates ◽  
Single Form ◽  
Half Site

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

scholarly journals Synchrotron radiation macromolecular crystallography: our science and spin offs

2014 ◽  
Vol 70 (a1) ◽  
pp. C10-C10
Author(s):  
John Helliwell
Keyword(s):  
Synchrotron Radiation ◽  
Protein Complexes ◽  
Bound Water ◽  
Water Structure ◽  
Public Understanding ◽  
Anomalous Scattering ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
Chemical Crystallography

I will give an overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications from the early days to the present, including the evolution of SR sources and on to the `ultimate storage ring'. The build of dedicated beamlines for resonant anomalous scattering, large unit cells, ever smaller crystals and studies up to ultra-high resolution are core benefits. Results include a high output of PDB depositions, the successful use of microcrystals, pushing the frontiers of using high and low photon energies and time-resolved structural studies at even sub-nanosecond resolutions. These intensively physics based developments will be complemented by biological and chemical crystallography research results, encompassing catalysis and marine coloration, as well as the public understanding of our science and its impacts. Spin off benefits include services to the pharmaceutical industry and helping develop chemical crystallography uses of SR. The development of the Laue method with SR has led to pioneering spin off developments in neutron MX, including transfer of the well validated Daresbury Laue software to various neutron facilities worldwide. Neutron MX is gathering pace as new instrumentation and dedicated sample preparation facilities are in place at reactor and spallation neutron sources; smaller samples and much larger molecular weight protein complexes are now feasible for investigation so as to establish their protonation states and bound water structure. With the X-ray lasers, closely linked to the SR developments, we anticipate the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules, as well as opening up femtosecond time-resolved diffraction structural studies. At the SR sources, a very high throughput assessment for the best crystal samples and tackling sub-micron crystals will become widespread.

scholarly journals The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

2019 ◽  
Author(s):  
Jifu Duan ◽  
Stefan Mebs ◽  
Moritz Senger ◽  
Konstantin Laun ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Active Site ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Hydrogen Bonding Interactions ◽  
Catalytic Active Site ◽  
Catalytic Intermediates ◽  
Time Resolved Infrared Spectroscopy

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.<br>

scholarly journals The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

2019 ◽  
Author(s):  
Jifu Duan ◽  
Stefan Mebs ◽  
Moritz Senger ◽  
Konstantin Laun ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Active Site ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Hydrogen Bonding Interactions ◽  
Catalytic Active Site ◽  
Catalytic Intermediates ◽  
Time Resolved Infrared Spectroscopy

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.<br>

Time-resolved phosphorescence measurement of conformational transitions in cation transport Na+, K+-ATPase

1993 ◽  
Author(s):  
Liqun Yang ◽  
Olga Lopina ◽  
Daniel McStay ◽  
A. A. Boldyrev ◽  
Alan J. Rogers ◽  
...  
Keyword(s):  
Cation Transport ◽  
Conformational Transitions ◽  
Time Resolved

scholarly journals Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

2020 ◽  
Vol 117 (33) ◽  
pp. 19720-19730 ◽  
Author(s):  
Yifan Wang ◽  
Kathy Fange Liu ◽  
Yu Yang ◽  
Ian Davis ◽  
Aimin Liu
Keyword(s):  
Conformational Changes ◽  
Quinolinic Acid ◽  
De Novo ◽  
Crystalline Lens ◽  
Paramagnetic Resonance ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Protein Conformational Changes ◽  
Catalytic Intermediates ◽  
Electron Paramagnetic

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolvedin crystalloreactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

scholarly journals Hazard and risk assessment strategies for nanoparticle exposures: how far have we come in the past 10 years?

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 376 ◽  
Author(s):  
David B Warheit
Keyword(s):  
Health Effects ◽  
Ultrafine Particles ◽  
Time Course ◽  
The European Union ◽  
Particle Size Range ◽  
Potential Health ◽  
The Past ◽  
Safety Research ◽  
Made In

Nanotechnology is an emerging, cross-disciplinary technology designed to create and synthesize new materials at the nanoscale (generally defined as a particle size range of ≤10-9 meters) to generate innovative or altered material properties. The particle properties can be modified to promote different and more flexible applications, resulting in consumer benefits, particularly in medical, cosmetic, and industrial applications. As this applied science matures and flourishes, concerns have arisen regarding potential health effects of exposures to untested materials, as many newly developed products have not been adequately evaluated. Indeed, it is necessary to ensure that societal and commercial advantages are not outweighed by potential human health or environmental disadvantages. Therefore, a variety of international planning activities or research efforts have been proposed or implemented, particularly in the European Union and United States, with the expectation that significant advances will be made in understanding potential hazards related to exposures in the occupational and/or consumer environments. One of the first conclusions reached regarding hazardous effects of nanoparticles stemmed from the findings of early pulmonary toxicology studies, suggesting that lung exposures to ultrafine particles were more toxic than those to larger, fine-sized particles of similar chemistry. This review documents some of the conceptual planning efforts, implementation strategies/activities, and research accomplishments over the past 10 years or so. It also highlights (in this author’s opinion) some shortcomings in the research efforts and accomplishments over the same duration. In general, much progress has been made in developing and implementing environmental, health, and safety research-based protocols for addressing nanosafety issues. However, challenges remain in adequately investigating health effects given 1) many different nanomaterial types, 2) various potential routes of exposure, 3) nanomaterial characterization issues, 4) limitations in research methodologies, such as time-course and dose-response issues, and 5) inadequate in vitro methodologies for in vivo standardized, guideline toxicity testing.

scholarly journals Approach of Serial Crystallography

Crystals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 854
Author(s):  
Ki Hyun Nam
Keyword(s):  
Radiation Damage ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Wide Range ◽  
Experimental Limitation ◽  
Serial Crystallography ◽  
Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

P5452First data-analysis of the prospective ETiCS-study after study-end confirms acute (microbial-induced) inflammation as a key trigger for the development of cardiac GPCR-autoantibodies

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
V Boivin ◽  
C Zechmeister ◽  
C Schuetz ◽  
N Beyersdorf ◽  
D Berliner ◽  
...  
Keyword(s):  
Time Course ◽  
Cardiovascular Death ◽  
Beta Blockade ◽  
Myocardial Inflammation ◽  
University Hospitals ◽  
Mortality And Morbidity ◽  
The Past ◽  
Experimental Therapies ◽  
Membrane Antigens ◽  
Cardiac Necrosis

Abstract Heart failure (HF) is the leading cause of mortality and morbidity in Western countries. In the past two decades, evidence for the clinical relevance of GPCR-autoimmunity in human HF has substantially increased. Stimulating autoantibodies targeting the second extracellular loop (ECII) of the cardiac beta1-adrenoceptor (beta1-aabs) have been claimed to be involved in the pathogenesis of HF and to increase the risk of cardiovascular death by three-fold. Still, the events triggering the formation of beta1-aabs and their impact on HF-progression are unknown. Methods In total 13 University Hospitals (12 German, 1 Serbian) prospectively recruited 226 patients (pts.) with a first acute myocardial infarction (FAMI), and 140 pts with acute (biopsy- or cMRI-proven) myocarditis (AMitis) into the Etiology, Titer-Course and effect on Survival of cardiac autoantibodies-study (ETiCS-study). This study aimed to investigate whether the presentation of cardiac membrane antigens (e.g., the beta1-adrenoceptor) following cardiac necrosis/inflammation triggers the formation of beta1-aabs. At baseline (BL) and three follow-ups (Fup1–3), blood was sampled to analyze the time-course of beta1-aabs. Beta1-aab titers were measured by FACS using Dyna-beads® M-270-Epoxy coated with increasing amounts of beta1-ECII-peptides (2.5–100 μg/ml), checked versus scrambled peptides (a mixture of same amino-acids). After reacting, the samples were measured by FACScan flow-cytometry; obtained data were analyzed with FlowJo (Treestar). When half-maximal binding was calculable the serum was classified beta1-aab-positive. Results From n=366 pts (226 FAMI/140 AMitis) recruited into the ETiCS-study 45 pts had to be excluded because of unperformed cMRI's; 46 pts stopped the study before Fup-1 (month 3). Only 180/226 FAMI- and 98/140 AMitis-pts had complete Fup1–3 (after 3, 6, and 12 months with clinical assessment, echocardiograms, and cMRI's at BL and Fup-3). In all valid ETiCS-pts (197 FAMI-/123 AMitis-pts) the titer-course of beta1-aabs was compared with the development of echo-LVEF. Relevant (high-affinity) beta1-aab-titers were detected in ∼31% (37/123) of the AMitis-pts compared to only ∼21% (42/197) of the FAMI-pts. In aab-positive AMitis-pts echo-LVEF did not recover and was always significantly inferior to aab-negative AMitis-pts (BL: 38 vs. 49% LVEF; Fup-3: 49 vs. 64% LVEF) whereas such a difference was not noted in FAMI-pts. In addition, aab-positive AMitis-pts had higher NT pro-BNP-, renin-, and aldosterone-levels than aab-negative AMitis-pts. Conclusion The first evaluation of the completed ETiCS-study clearly suggests that acute microbial-induced rather than post-infarction myocardial inflammation triggers the formation of clinically relevant beta1-aabs. AAb-positive AMitis-patients might profit from early intensification of standard HF-therapy (including early beta-blockade) and/or novel antibody-directed experimental therapies which are currently developed. Acknowledgement/Funding BMBF Grant FKZ 01ES0816

scholarly journals Two Quenchers Formed During Photodamage of Phostosystem II and The Role of One Quencher in Preemptive Photoprotection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alonso Zavafer ◽  
Ievgeniia Iermak ◽  
Mun Hon Cheah ◽  
Wah Soon Chow
Keyword(s):  
Chlorophyll Fluorescence ◽  
Photosystem Ii ◽  
Time Course ◽  
Physiological Role ◽  
Reaction Centre ◽  
Time Resolved ◽  
Fluorescence Quencher

AbstractThe quenching of chlorophyll fluorescence caused by photodamage of Photosystem II (qI) is a well recognized phenomenon, where the nature and physiological role of which are still debatable. Paradoxically, photodamage to the reaction centre of Photosystem II is supposed to be alleviated by excitation quenching mechanisms which manifest as fluorescence quenchers. Here we investigated the time course of PSII photodamage in vivo and in vitro and that of picosecond time-resolved chlorophyll fluorescence (quencher formation). Two long-lived fluorescence quenching processes during photodamage were observed and were formed at different speeds. The slow-developing quenching process exhibited a time course similar to that of the accumulation of photodamaged PSII, while the fast-developing process took place faster than the light-induced PSII damage. We attribute the slow process to the accumulation of photodamaged PSII and the fast process to an independent quenching mechanism that precedes PSII photodamage and that alleviates the inactivation of the PSII reaction centre.

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