Osmolyte-induced protein folding free energy changes

Area of Science:

• Biochemistry
• Chemical Thermodynamics
• Protein Folding

Background:

• Spectral changes in intrinsically unstructured proteins are observed with increasing osmolyte concentration.
• The linear extrapolation model (LEM) is commonly used to analyze protein folding transitions.
• Accurate definition of the 0%-100% folding scale is crucial for LEM analysis.

Purpose of the Study:

• To determine the thermodynamic stability of reduced and carboxyamidated RNase T1 (RCAM-T1) induced by osmolytes.
• To address inaccuracies in the conventional LEM arising from non-zero folded protein fractions in buffer alone.
• To validate a corrected LEM for deriving authentic thermodynamic parameters of protein folding.

Main Methods:

• Utilized reduced and carboxyamidated RNase T1 (RCAM-T1) as an intrinsically unstructured protein model.
• Employed urea titration and fluorescence spectroscopy to establish a corrected denatured-state baseline (F/F0extrap = 1.0).
• Applied the corrected LEM to calculate thermodynamic stability (ΔG[D-->N0]) in the presence of various osmolytes.

Main Results:

• The conventional LEM yielded inaccurate ΔG[D-->N0] values due to an assumption of complete unfolding in buffer.
• The corrected LEM produced identical and precise ΔG[D-->N0] values for RCAM-T1 folding across different osmolytes.
• The rank order of osmolyte efficacy in stabilizing RCAM-T1 was determined: sarcosine > sucrose > sorbitol > proline > betaine > glycerol.

Conclusions:

• The corrected LEM provides a robust method for accurately determining protein folding thermodynamic stability.
• The derived ΔG[D-->N0] values are independent of the specific osmolytes used, supporting their thermodynamic validity.
• This study validates the LEM as a reliable tool for quantifying protein stabilization by osmolytes.