Conformational Landscapes Observed through Controlled Kinetics (CLOCK): Precise Control of Ion Temperatures from...

Area of Science:

• Structural mass spectrometry
• Ion mobility spectrometry
• Biophysical characterization

Background:

• Understanding ion conformational dynamics across temperatures and timescales is crucial in structural mass spectrometry.
• Traveling-wave ion mobility (IM) instruments built on structures for lossless ion manipulations (SLIM) offer potential for such studies.
• Controlled kinetics (CLOCK) was previously introduced as a proof-of-concept for observing conformational landscapes.

Purpose of the Study:

• To develop controlled kinetics (CLOCK) into a quantitative framework for probing ion dynamics on submillisecond to second timescales.
• To investigate the influence of activation parameters on ion conformational distributions.
• To establish a temperature-resolved and time-resolved method for analyzing ion dynamics.

Main Methods:

• Application of the CLOCK framework to native-like ions of 6+ ubiquitin and 7+ cytochrome c.
• Independent control and analysis of guard electrode potential and activation duration.
• Combination of ion trajectory simulations with statistical methods to estimate effective translational temperatures.
• Quantitative analysis of ion unfolding kinetics using the calibrated CLOCK framework.

Main Results:

• Both guard electrode potential and activation duration independently control conformational distributions of ubiquitin and cytochrome c ions.
• Effective translational temperatures were estimated, providing a physically interpretable scale for activation conditions.
• CLOCK experiments, calibrated by temperature, revealed distinct unfolding kinetics for 6+ ubiquitin and 7+ cytochrome c over similar effective temperature ranges.

Conclusions:

• CLOCK is established as a quantitative, temperature-resolved, and time-resolved framework for probing ion conformational dynamics.
• The study provides mechanistic insights into CLOCK and its application in structural mass spectrometry.
• Distinct ion unfolding behaviors were observed, highlighting the utility of CLOCK for comparative biophysical studies.