scholarly journals The Use of the Time-Resolved X-Ray Solution Scattering for Studies of Globular Proteins

2002 ◽  
Vol 16 (3-4) ◽  
pp. 127-138 ◽  
Author(s):  
Kunihiro Kuwajima ◽  
Munehito Arai ◽  
Tomonao Inobe ◽  
Kazuki Ito ◽  
Masaharu Nakao ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Early Stage ◽  
Molten Globule ◽  
Image Intensifier ◽  
Image Sensor ◽  
Spectroscopic Techniques ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

In order to improve the low signal-to-noise ratio of the time-resolved small-angle X-ray scattering, we have used a two-dimensional X-ray detector with a beryllium-windowed X-ray image intensifier and a charge-coupled device as an image sensor, and applied this to studies on (1) the kinetic folding reaction of α-lactalbumin, which accumulates the molten globule-like intermediate at an early stage of refolding and (2) the cooperative conformational transition ofEscherichia colichaperonin GroEL induced by ATP, which occurs in an allosteric manner between the close and open conformational states. In the α-lactalbumin reaction, we have firmly established the equivalence between the kinetic intermediate and the equilibrium molten globule state, and obtained further information about dehydration from the highly hydrated folding intermediate during a late stage of refolding. In the chaperonin study, we have successfully observed the kinetics of the allosteric transition of GroEL that occurs with a rate constant of about 3–4 s−1at 5°C. The combination of the time-resolved X-ray scattering with other spectroscopic techniques such as circular dichroism and intrinsic fluorescence is thus very effective in understanding the conformational transitions of proteins and protein complexes.

scholarly journals Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

2018 ◽  
Author(s):  
Michael C. Thompson ◽  
Benjamin A. Barad ◽  
Alexander M. Wolff ◽  
Hyun Sun Cho ◽  
Friedrich Schotte ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Conformational Changes ◽  
Thermal Excitation ◽  
Relaxation Processes ◽  
Spectroscopic Techniques ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Solvent Molecules ◽  
Ray Scattering

AbstractCorrelated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the μs-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

Time-Resolved Small-Angle X-ray Scattering Study of Reversion Kinetics of δ′ Precipitates in an Al - 12AT.% Li Alloy Utilizing Synchrotron Radiation

1990 ◽  
Vol 205 ◽  
Author(s):  
Haydn Chen ◽  
M.S. Yu ◽  
H. Okuda ◽  
M. Tanaka ◽  
K. Osamura
Keyword(s):  
Synchrotron Radiation ◽  
Small Angle ◽  
Early Stage ◽  
Radius Of Gyration ◽  
Second Phase ◽  
Time Resolved ◽  
X Ray ◽  
Diffuse Interfaces ◽  
X Ray Scattering ◽  
Ray Scattering

AbstractStructure change during the reversion process in an Al−12at.%Li alloy above the metastable δ′ solvus was investigated using a time-resolved small-angle x-ray scattering technique with synchrotron radiation. Results showed that the reversion process started after a short incubation time and that the growth of the stable δ phase began before completion of the δ′ dissolution. The radius of gyration of the second phase particles showed little change in the initial stage of reversion, then increased with time, suggesting the presence of diffuse interfaces between the dissolving δ′ particles and the matrix. It is suggested that the undissolved δ′ particles serve as the nuclei of the more stable δ precipitates, which continue to grow with their radii of gyration showing a parabolic power law in the early stage of growth followed by the familiar coarsening kinetics.

Time-resolved small-angle x-ray scattering study of the early stage of amyloid formation of an apomyoglobin mutant

2011 ◽  
Vol 84 (6) ◽  
Author(s):  
Maria Grazia Ortore ◽  
Francesco Spinozzi ◽  
Silvia Vilasi ◽  
Ivana Sirangelo ◽  
Gaetano Irace ◽  
...  
Keyword(s):  
Small Angle ◽  
Early Stage ◽  
Amyloid Formation ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Study ◽  
Ray Scattering

scholarly journals Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering

Polymers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2104
Author(s):  
Yecheol Rho ◽  
Jun Ha Kim ◽  
Byoungseok Min ◽  
Kyeong Sik Jin
Keyword(s):  
Small Angle ◽  
Conformational Transition ◽  
Early Stage ◽  
3D Structure ◽  
Three Dimensional ◽  
Porcine Pepsin ◽  
X Ray ◽  
X Ray Scattering ◽  
Wide Range ◽  
Ray Scattering

Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0–10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0–5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7–10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.

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?

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.

Time-Resolved, in Situ, Small- and Wide-Angle X-ray Scattering To Monitor Pt Nanoparticle Structure Evolution Stabilized by Adsorbed SnCl3– Ligands During Synthesis

2013 ◽  
Vol 117 (15) ◽  
pp. 7924-7933 ◽  
Author(s):  
Samuel St. John ◽  
Naiping Hu ◽  
Dale W. Schaefer ◽  
Anastasios P. Angelopoulos
Keyword(s):  
Structure Evolution ◽  
Wide Angle ◽  
Pt Nanoparticle ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Nanoparticle Structure ◽  
Ray Scattering

scholarly journals Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study

2019 ◽  
Vol 6 (3) ◽  
pp. 034701 ◽  
Author(s):  
Saskia Bannister ◽  
Elena Böhm ◽  
Thomas Zinn ◽  
Thomas Hellweg ◽  
Tilman Kottke
Keyword(s):  
Small Angle ◽  
Full Length ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Study ◽  
Ray Scattering
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