Dataset Information

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin.

ABSTRACT: Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with nonlinear IR spectroscopy of the amide I vibrations. Equilibrium temperature-dependent 2D IR spectroscopy reveals the unfolding of the beta-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from delocalized vibrations of the beta-sheet. Transient unfolding after a nanosecond temperature jump is monitored with dispersed vibrational echo spectroscopy, a projection of the 2D IR spectrum. Whereas the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans 6 decades in time. The transient behavior can be separated into fast and slow time scales. From 100 ns to 0.5 ms, the spectral features associated with beta-sheet unfolding relax in a sequential, nonexponential manner, with time constants of 3 micros and 80 micros. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands III-V of the beta-sheet before unfolding of the hairpin that forms part of the hydrophobic core. This downhill unfolding is followed by exponential barrier-crossing kinetics on a 3-ms time scale.

SUBMITTER: Chung HS

PROVIDER: S-EPMC545570 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature

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Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin.

Chung Hoi Sung HS Khalil Munira M Smith Adam W AW Ganim Ziad Z Tokmakoff Andrei A

Proceedings of the National Academy of Sciences of the United States of America 20050103 3

Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with nonlinear IR spectroscopy of the amide I vibrations. Equilibrium temperature-dependent 2D IR spectroscopy reveals the unfolding of the beta-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from delocalized vibrations of the beta-sheet. Transient unfolding after a nanosecond temperature jump is monitored with dispersed vibrational echo spectroscopy, ...[more]

PMID: 15630083

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Project description:Nanosecond Pulsed Electric Field (nsPEF or Nano Pulsed Stimulation, NPS) is a technology that delivers a series of pulses of high-voltage electric fields during a short period of time, in the order of nanoseconds. The main consequence of nsPEF upon cells is the formation of nanopores, which is followed by the gating of ionic channels. Literature is conclusive in that the physiological mechanisms governing ion channel gating occur in the order of milliseconds. Hence, understanding how these channels can be activated by a nsPEF would be an important step in order to conciliate fundamental biophysical knowledge with improved nsPEF applications. To get insights on both the kinetics and thermodynamics of ion channel gating induced by nsPEF, in this work, we simulated the Voltage Sensing Domain (VSD) of a voltage-gated Ca2+ channel, inserted in phospholipidic membranes with different concentrations of cholesterol. We studied the conformational changes of the VSD under a nsPEF mimicked by the application of a continuous electric field lasting 50 ns with different intensities as an approach to reveal novel mechanisms leading to ion channel gating in such short timescales. Our results show that using a membrane with high cholesterol content, under an nsPEF of 50 ns and E→ = 0.2 V/nm, the VSD undergoes major conformational changes. As a whole, our work supports the notion that membrane composition may act as an allosteric regulator, specifically cholesterol content, which is fundamental for the response of the VSD to an external electric field. Moreover, changes on the VSD structure suggest that the gating of voltage-gated Ca2+ channels by a nsPEF may be due to major conformational changes elicited in response to the external electric field. Finally, the VSD/cholesterol-bilayer under an nsPEF of 50 ns and E→ = 0.2 V/nm elicits a pore formation across the VSD suggesting a new non-reported effect of nsPEF into cells, which can be called a "protein mediated electroporation".

Dataset Information

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin.

Publications

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin.

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