A kinetic folding intermediate probed by native state hydrogen exchange

Area of Science:

• Protein folding dynamics
• Biophysical chemistry
• Structural biology

Background:

• The N-terminal domain of rat CD2 (CD2.d1) folding involves a burst phase with a transient intermediate (I) preceding the native state (N).
• Previous hydrogen exchange (HX) studies suggested that certain amides exchange from the native state under EX1 conditions, indicating involvement of the kinetic transition state barrier.

Purpose of the Study:

• To investigate the structural and energetic properties of kinetic folding intermediates in CD2.d1 using native state HX (NHX) under varying denaturant concentrations.
• To confirm the utility of NHX for characterizing transient folding intermediates.

Main Methods:

• Stopped-flow fluorescence spectroscopy to study protein folding kinetics.
• pH-dependent equilibrium hydrogen exchange (HX) studies under EX1 and EX2 conditions.
• Native state HX (NHX) measurements across a range of denaturant concentrations.

Main Results:

• NHX data fit a two-component (sub-global/global) mechanism, yielding thermodynamic parameters (ΔG, m-value).
• Regression analysis showed correspondence between calculated (m, ΔG) values for amides and the kinetic burst phase transition.
• This confirms that NHX can probe the structural and energetic characteristics of kinetic folding intermediates.

Conclusions:

• Native state hydrogen exchange is a powerful technique for characterizing the structural and energetic landscape of transient protein folding intermediates.
• The study validates the NHX method for exploring the kinetic burst phase of protein folding.