Carbon−Deuterium Bonds as Site-Specific and Nonperturbative Probes for Time-Resolved Studies of Protein Dynamics and Folding

2011 ◽  
Vol 2 (5) ◽  
pp. 412-416 ◽  
Author(s):  
Jörg Zimmermann ◽  
Megan C. Thielges ◽  
Wayne Yu ◽  
Philip E. Dawson ◽  
Floyd E. Romesberg
Keyword(s):  
Protein Dynamics ◽  
Time Resolved ◽  
Site Specific

scholarly journals Site-specific Protein Dynamics in Communication Pathway from Sensor to Signaling Domain of Oxygen Sensor Protein, HemAT-Bs

2012 ◽  
Vol 287 (24) ◽  
pp. 19973-19984 ◽  
Author(s):  
Samir F. El-Mashtoly ◽  
Minoru Kubo ◽  
Yuzong Gu ◽  
Hitomi Sawai ◽  
Satoru Nakashima ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Oxygen Sensor ◽  
Specific Protein ◽  
Site Specific ◽  
Sensor Protein ◽  
Signaling Domain ◽  
Communication Pathway

Site-Specific Mapping and Time-Resolved Monitoring of Lysine Methylation by High-Resolution NMR Spectroscopy

2012 ◽  
Vol 134 (18) ◽  
pp. 7616-7619 ◽  
Author(s):  
François-Xavier Theillet ◽  
Stamatios Liokatis ◽  
Jan Oliver Jost ◽  
Beata Bekei ◽  
Honor May Rose ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
High Resolution ◽  
Lysine Methylation ◽  
Time Resolved ◽  
Site Specific ◽  
High Resolution Nmr ◽  
Specific Mapping

scholarly journals A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

IUCrJ ◽  
2016 ◽  
Vol 3 (6) ◽  
pp. 393-401 ◽  
Author(s):  
Robin Schubert ◽  
Svetlana Kapis ◽  
Yannig Gicquel ◽  
Gleb Bourenkov ◽  
Thomas R. Schneider ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Temporal Resolution ◽  
Diffraction Data ◽  
Structural Changes ◽  
Structural Information ◽  
Data Sets ◽  
Enzymatic Reactions ◽  
Time Resolved ◽  
Site Specific

Many biochemical processes take place on timescales ranging from femtoseconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach forin situcrystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

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.

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