Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis

Nature ◽  
1990 ◽  
Vol 345 (6273) ◽  
pp. 309-315 ◽  
Author(s):  
Ilme Schlichting ◽  
Steven C. Almo ◽  
Gert Rapp ◽  
Keith Wilson ◽  
Kyriakos Petratos ◽  
...  
Keyword(s):  
Conformational Change ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
P21 Protein ◽  
X Ray ◽  
Crystallographic Study ◽  
Ras P21

The Hydration of Ras p21 in Solution during GTP Hydrolysis Based on Solution X-Ray Scattering Profile1

1994 ◽  
Vol 115 (5) ◽  
pp. 875-880 ◽  
Author(s):  
Tetsuro Fujisawa ◽  
Tomoya Uruga ◽  
Ziro Yamaizumi ◽  
Yoji Inoko ◽  
Susumu Nishimura ◽  
...  
Keyword(s):  
Gtp Hydrolysis ◽  
X Ray ◽  
X Ray Scattering ◽  
Ras P21 ◽  
Ray Scattering

scholarly journals Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

2012 ◽  
Vol 109 (38) ◽  
pp. 15295-15300 ◽  
Author(s):  
Till Rudack ◽  
Fei Xia ◽  
Jürgen Schlitter ◽  
Carsten Kötting ◽  
Klaus Gerwert
Keyword(s):  
Nucleophilic Attack ◽  
Nonbridging Oxygen ◽  
Difference Spectra ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
X Ray ◽  
Cellular Processes ◽  
Gtpase Activating Proteins ◽  
Biomolecular Simulations ◽  
Oxygen Atoms

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and Pi by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 105 compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 105 compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

scholarly journals Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein

2016 ◽  
Vol 18 (13) ◽  
pp. 8911-8919 ◽  
Author(s):  
Tae Wu Kim ◽  
Cheolhee Yang ◽  
Youngmin Kim ◽  
Jong Goo Kim ◽  
Jeongho Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Conformational Change ◽  
Absorption Spectroscopy ◽  
Structural Changes ◽  
Transient Absorption ◽  
Transient Absorption Spectroscopy ◽  
Photoactive Yellow Protein ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering

Structural changes in global conformation and chromophore of photoactive yellow protein are probed in real time by time-resolved X-ray solution scattering and transient absorption spectroscopy.

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 766-767
Author(s):  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
Joan Bordas
Keyword(s):  
Electron Microscopy ◽  
Dynamic Equilibrium ◽  
Time Resolved ◽  
X Ray ◽  
Additional Information ◽  
X Ray Scattering ◽  
Low Concentrations ◽  
Microtubule Growth ◽  
Ray Patterns ◽  
Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

Time-resolved X-ray reciprocal space mapping of the crystal under external electric field

2018 ◽  
Vol 189 (02) ◽  
pp. 187-194 ◽  
Author(s):  
Nikita V. Marchenkov ◽  
Anton G. Kulikov ◽  
Ivan I. Atknin ◽  
Arsen A. Petrenko ◽  
Alexander E. Blagov ◽  
...  
Keyword(s):  
Electric Field ◽  
External Electric Field ◽  
Space Mapping ◽  
Reciprocal Space ◽  
Reciprocal Space Mapping ◽  
Time Resolved ◽  
X Ray

Monitoring Polymer-Assisted Mechanochemical Cocrystallisation Through in Situ X-Ray Powder Diffraction

2020 ◽  
Author(s):  
Luzia S. Germann ◽  
Sebastian T. Emmerling ◽  
Manuel Wilke ◽  
Robert E. Dinnebier ◽  
Mariarosa Moneghini ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Powder Diffraction ◽  
Reaction Rate ◽  
Polymer Additives ◽  
Time Resolved ◽  
X Ray

Time-resolved mechanochemical cocrystallisation studies have so-far focused solely on neat and liquid-assisted grinding. Here, we report the monitoring of polymer-assisted grinding reactions using <i>in situ</i> X-ray powder diffraction, revealing that reaction rate is almost double compared to neat grinding and independent of the molecular weight and amount of used polymer additives.<br>

In situ Time-Resolved Small-Angle X-ray Scattering Reveals the Formation Kinetics of Polyelectrolyte Complex Micelles

2019 ◽  
Author(s):  
Hao Wu ◽  
Jeffrey Ting ◽  
Siqi Meng ◽  
Matthew Tirrell
Keyword(s):  
Small Angle ◽  
Self Assembly ◽  
Electrostatic Interactions ◽  
Polyelectrolyte Complex ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Kinetics Of ◽  
Ray Scattering

We have directly observed the <i>in situ</i> self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. This work has elucidated one general kinetic pathway for the process of PEC micelle formation, which provides useful physical insights for increasing our fundamental understanding of complexation and self-assembly dynamics driven by electrostatic interactions that occur on ultrafast timescales.

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