scholarly journals Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering

2020 ◽  
Vol 6 (12) ◽  
pp. eaaz0981 ◽  
Author(s):  
Harsha Ravishankar ◽  
Martin Nors Pedersen ◽  
Mattias Eklund ◽  
Aljona Sitsel ◽  
Chenge Li ◽  
...  
Keyword(s):  
Real Time ◽  
Transient State ◽  
Md Simulations ◽  
Relative Timing ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Active Calcium ◽  
Real Time Tracking ◽  
Domain Arrangement

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

scholarly journals Real-time tracking of protein unfolding with time-resolved x-ray solution scattering

2020 ◽  
Vol 7 (5) ◽  
pp. 054702
Author(s):  
L. Henry ◽  
M. R. Panman ◽  
L. Isaksson ◽  
E. Claesson ◽  
I. Kosheleva ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Unfolding ◽  
Time Resolved ◽  
X Ray ◽  
Real Time Tracking

Picosecond time-resolved X-ray crystallography: probing protein function in real time

2004 ◽  
Vol 147 (3) ◽  
pp. 235-246 ◽  
Author(s):  
Friedrich Schotte ◽  
Jayashree Soman ◽  
John S. Olson ◽  
Michael Wulff ◽  
Philip A. Anfinrud
Keyword(s):  
Real Time ◽  
Protein Function ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography

Can Laue catch Maxwell?: observation of short-lived species by Laue X-ray crystallography

1992 ◽  
Vol 340 (1657) ◽  
pp. 273-284 ◽  
Keyword(s):  
Real Time ◽  
Protein Crystal ◽  
Separate Experiment ◽  
Enzymatic Reactions ◽  
X Ray Diffraction ◽  
Single Experiment ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Novel Strategy

Now that the Laue method has been established as a tool for protein crystallography, the main problem involved in any prospective time-resolved X-ray diffraction study is one of chemistry. The reaction or process in question must be initiated on a timescale that is fast compared with its kinetics. For most biochemical events photochemistry is the most suitable trigger, but not all substrates can be caged for photochemical release. This problem can be solved by the novel strategy of caging the enzyme with a photoreversible covalent inhibitor. The logic of this method will be discussed and its application to a time-resolved study of the reaction of a suicide substrate with the protease gamma chymotrypsin shown. The question of real-time crystallographic ‘movies’ of enzymatic reactions can now be considered. It seems likely that following a reaction in real time in a single experiment will be very difficult if not impossible in most cases, in part because even a synchronized process will rapidly become asynchronous in a protein crystal, and also because it will be very difficult to know exactly what species one is observing at any instant unless one has extremely high resolution. It seems that the best use of the Laue technique will be to study unstable species that can be accumulated in the crystal under defined conditions for short periods of time. An entire reaction sequence can then be obtained as a series of individual steps, each of which is obtained from a separate experiment.

A flow cell suitable for time-resolved X-ray crystallography by the Laue method

1997 ◽  
Vol 30 (5) ◽  
pp. 555-556 ◽  
Author(s):  
G. Kurisu ◽  
A. Sugimoto ◽  
Y. Kai ◽  
S. Harada
Keyword(s):  
Flow Cell ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Laue Method

scholarly journals Probing G-quadruplex topologies and recognition concurrently in real time and 3D using a dual-app nucleoside probe

2019 ◽  
Vol 47 (12) ◽  
pp. 6059-6072 ◽  
Author(s):  
Ashok Nuthanakanti ◽  
Ishtiyaq Ahmed ◽  
Saddam Y Khatik ◽  
Kayarat Saikrishnan ◽  
Seergazhi G Srivatsan
Keyword(s):  
Nucleic Acid ◽  
Ligand Binding ◽  
Real Time ◽  
Telomeric Dna ◽  
X Ray ◽  
X Ray Crystallography ◽  
New Class ◽  
Loop Region ◽  
Dna Repeat ◽  
G Quadruplex

Abstract Comprehensive understanding of structure and recognition properties of regulatory nucleic acid elements in real time and atomic level is highly important to devise efficient therapeutic strategies. Here, we report the establishment of an innovative biophysical platform using a dual-app nucleoside analog, which serves as a common probe to detect and correlate different GQ structures and ligand binding under equilibrium conditions and in 3D by fluorescence and X-ray crystallography techniques. The probe (SedU) is composed of a microenvironment-sensitive fluorophore and an excellent anomalous X-ray scatterer (Se), which is assembled by attaching a selenophene ring at 5-position of 2′-deoxyuridine. SedU incorporated into the loop region of human telomeric DNA repeat fluorescently distinguished subtle differences in GQ topologies and enabled quantify ligand binding to different topologies. Importantly, anomalous X-ray dispersion signal from Se could be used to determine the structure of GQs. As the probe is minimally perturbing, a direct comparison of fluorescence data and crystal structures provided structural insights on how the probe senses different GQ conformations without affecting the native fold. Taken together, our dual-app probe represents a new class of tool that opens up new experimental strategies to concurrently investigate nucleic acid structure and recognition in real time and 3D.

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