Understanding cold denaturation: the case study of Yfh1

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Globular proteins transition between native and unfolded states due to heat or cold.
• Heat-induced protein denaturation is well-understood, but cold denaturation is less characterized, often requiring artificial destabilization.
• Understanding cold denaturation is crucial for biological processes in cold environments.

Purpose of the Study:

• To characterize the low-temperature unfolded state of Yfh1, a natural protein exhibiting cold denaturation near water freezing temperature.
• To investigate the structural and dynamic features of Yfh1 at -1 °C in the absence of denaturants.
• To provide the first detailed characterization of a naturally cold-denatured protein state.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy for near-complete spectral assignment.
• Analysis of protein conformational changes at low temperatures.
• Characterization of residual secondary structure and dynamic flexibility.

Main Results:

• At -1 °C, Yfh1 exhibits characteristics of an unfolded protein, with some residual local secondary structure.
• The unfolding is sequence-non-uniform, with the N-terminus showing increased flexibility and nascent helix character, containing functional hotspots.
• The β-sheet region and C-terminal helix are fully unfolded, with some conformational exchange influenced by proline residues.

Conclusions:

• Yfh1 undergoes natural cold denaturation without external agents.
• The low-temperature unfolded state is heterogeneous, with distinct regional dynamics.
• This study is a foundational step towards understanding natural cold-denatured protein states and their implications.