scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

scholarly journals A beginner’s guide to macromolecular crystallization

2021 ◽  
Vol 43 (1) ◽  
pp. 36-43
Author(s):  
Fabrice Gorrec
Keyword(s):  
Crystal Nucleation ◽  
Trial And Error ◽  
Bioinformatics Analyses ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

scholarly journals Iterative screen optimization maximizes the efficiency of macromolecular crystallization

2019 ◽  
Vol 75 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Harrison G. Jones ◽  
Daniel Wrapp ◽  
Morgan S. A. Gilman ◽  
Michael B. Battles ◽  
Nianshuang Wang ◽  
...  
Keyword(s):  
Narrow Range ◽  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Experiment ◽  
Automated Process ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations.

Oxidation of L-tryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase

2009 ◽  
Vol 37 (2) ◽  
pp. 408-412 ◽  
Author(s):  
Sara A. Rafice ◽  
Nishma Chauhan ◽  
Igor Efimov ◽  
Jaswir Basran ◽  
Emma Lloyd Raven
Keyword(s):  
Structural Information ◽  
Recombinant Expression ◽  
Kynurenine Pathway ◽  
Expression Systems ◽  
Indoleamine 2,3 Dioxygenase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
The Family ◽  
And Function ◽  
Rate Limiting

The family of haem dioxygenases catalyse the initial oxidative cleavage of L-tryptophan to N-formylkynurenine, which is the first, rate-limiting, step in the L-kynurenine pathway. In the present paper, we discuss and compare structure and function across the family of haem dioxygenases by focusing on TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase), including a review of recent structural information for both enzymes. The present paper describes how the recent development of recombinant expression systems has informed our more detailed understanding of the substrate binding, catalytic activity and mechanistic properties of these haem dioxygenases.

scholarly journals The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism

2015 ◽  
Vol 17 (46) ◽  
pp. 30793-30804 ◽  
Author(s):  
Katarzyna Świderek ◽  
Amnon Kohen ◽  
Vicent Moliner
Keyword(s):  
Reaction Mechanism ◽  
Thymidylate Synthase ◽  
Active Site ◽  
Hydride Transfer ◽  
Md Simulations ◽  
Concerted Mechanism ◽  
X Ray ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

QM/MM MD simulations from different X-ray structures support the concerted mechanism character in the rate limiting step of thymidylate synthase catalysis.

Formation and Decomposition of Nitrides on Iron Surfaces

1979 ◽  
Vol 34 (1) ◽  
pp. 30-39 ◽  
Author(s):  
G. Ertl ◽  
M. Huber ◽  
N. Thiele
Keyword(s):  
Photoelectron Spectroscopy ◽  
Electron Spectroscopy ◽  
Auger Electron ◽  
Thermal Desorption Spectroscopy ◽  
Iron Nitrides ◽  
Kcal Mole ◽  
X Ray ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract The formation (by interaction with ammonia) and decomposition of nitrides on clean Fe surfaces was studied by means of Auger electron spectroscopy, x-ray photoelectron spectroscopy, thermal desorption spectroscopy, and scanning electron microscopy. The N atoms may exist in various forms with quite similar electronic properties, viz. as chemisorbed layer (= "surface nitride"), dissolved in α-Fe or γ-Fe, as γ′-nitride (= Fe4N) or as e-nitride, depending on temperature as well as pressure and duration of interaction with NH3. There is no noticeable chemical shift of the ionization energies of the Fe core levels, indicating that the bond is essentially covalent. The activation energy for the decomposition of e-nitride into Fe4N + N2 is about 27 kcal/mole, that for the decomposition of Fe4N into Fe+N2 ranges between 51 and 57 kcal/mole, depending on the mode of preparation. The latter values are identical to those found previously for the desorption of N2 from various Fe single crystal planes and indicate that the decomposition of the chemisorbed "surface nitrides" is the rate-limiting step which prevents the spontaneous decom-position of the metastable bulk iron nitrides.

Myosin cross bridges in skeletal muscles: “rower” molecular motors

2001 ◽  
Vol 91 (6) ◽  
pp. 2479-2486 ◽  
Author(s):  
Y. Lecarpentier ◽  
D. Chemla ◽  
J. C. Pourny ◽  
F.-X. Blanc ◽  
C. Coirault
Keyword(s):  
Molecular Motors ◽  
Skeletal Muscles ◽  
Myosin Ii ◽  
Large Percentage ◽  
State Model ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Adp Release ◽  
Cross Bridges ◽  
Rate Limiting

Different classes of molecular motors, “rowers” and “porters,” have been proposed to describe the chemomechanical transduction of energy. Rowers work in large assemblies and spend a large percentage of time detached from their lattice substrate. Porters behave in the opposite way. We calculated the number of myosin II cross bridges (CB) and the probabilities of attached and detached states in a minimal four-state model in slow (soleus) and fast (diaphragm) mouse skeletal muscles. In both muscles, we found that the probability of CB being detached was ∼98% and the number of working CB was higher than 109/mm2. We concluded that muscular myosin II motors were classified in the category of rowers. Moreover, attachment time was higher than time stroke and time for ADP release. The duration of the transition from detached to attached states represented the rate-limiting step of the overall attached time. Thus diaphragm and soleus myosins belong to subtype 1 rowers.

scholarly journals Structural Characterization of Covalently Stabilized Human Cystatin C Oligomers

2020 ◽  
Vol 21 (16) ◽  
pp. 5860
Author(s):  
Magdalena Chrabąszczewska ◽  
Adam K. Sieradzan ◽  
Sylwia Rodziewicz-Motowidło ◽  
Anders Grubb ◽  
Christopher M. Dobson ◽  
...  
Keyword(s):  
Cystatin C ◽  
Protein Misfolding ◽  
Structural Information ◽  
Outer Edge ◽  
Cysteine Protease Inhibitor ◽  
X Ray ◽  
X Ray Crystallography ◽  
Human Cystatin C ◽  
Dynamics Simulations ◽  
Human Cystatin

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10–12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease.

scholarly journals Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca2+-ATPase

1995 ◽  
Vol 310 (3) ◽  
pp. 875-879 ◽  
Author(s):  
A P Starling ◽  
J M East ◽  
A G Lee
Keyword(s):  
Atp Hydrolysis ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Phosphoryl Transfer ◽  
Acyl Chain Length ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the Ca(2+)-ATPase purified from sarcoplasmic reticulum have been studied after reconstitution into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC], dioleoylphosphatidylcholine[di(C18:1)PC] and dinervonylphosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the rate of phosphorylation of the ATPase by ATP was comparable with that in di(C18:1)PC (about 70 s-1), but in di(C14:1)PC the rate was much lower (21 s-1). Fluorescence responses of the ATPase suggest changes in the phosphoryl-transfer step rather than in the preceding conformational change E1Ca2ATP<-->E1′Ca2ATP. The rate of dephosphorylation of the phosphorylated ATPase was found to decrease in the order di(C24:1)PC < di(C14:1)PC < di(C18:1)PC. For the ATPase in di(C24:1)PC the rate of dephosphorylation (3.3 s-1) was slow enough to be the rate-limiting step for ATP hydrolysis; in di(C14:1)PC, it is suggested that both phosphorylation and dephosphorylation contribute to rate limitation. Phosphorylation of the ATPase in di(C24:1)PC by Pi was normal, but no phosphoenzyme could be detected in di(C14:1)PC. The rate of the Ca(2+)-transport step was normal in di(C24:1)PC, suggesting that the single Ca2+ ion bound to the ATPase in di(C24:1)PC could be transported.

scholarly journals Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

2009 ◽  
Vol 20 (19) ◽  
pp. 4256-4266 ◽  
Author(s):  
Fu-Cheng Liang ◽  
Umesh K. Bageshwar ◽  
Siegfried M. Musser
Keyword(s):  
Protein Transport ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Precursor Proteins ◽  
Inverted Membrane Vesicles ◽  
Rate Limiting

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

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