Propensity for cis-Proline Formation in Unfolded Proteins.

Area of Science:

• Structural Biology
• Biochemistry
• Protein Folding Dynamics

Background:

• Peptide bonds involving proline (Pro) residues exist in cis and trans conformations, influencing protein folding.
• Cis-trans Pro isomerization is a known rate-limiting step in protein folding.
• Enzymes like kinases and phosphatases show differential recognition of cis- and trans-Pro conformers.

Purpose of the Study:

• To accurately determine the population of cis-Pro conformers in unfolded proteins, specifically the intrinsically disordered protein alpha-synuclein.
• To investigate the influence of experimental conditions, such as pressure and ionic strength, on cis-Pro populations.
• To compare cis-Pro populations in intrinsically disordered proteins with those in short model peptides.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was employed to analyze protein structures.
• Experiments were conducted under both atmospheric and high-pressure conditions.
• The intrinsically disordered protein alpha-synuclein and short peptides of similar sequences were studied.

Main Results:

• Cis-Pro populations in alpha-synuclein were found to be less than 5% for all five X-Pro bonds.
• Ionic strength showed only a modest effect on cis-Pro populations, and N-C terminal interactions had no detectable effect.
• Short peptides, especially those with charged termini, significantly overestimate the amount of cis-Pro compared to the protein.

Conclusions:

• Previous estimates of cis-Pro populations in unfolded proteins are likely overestimated due to the use of short peptides with charged termini.
• The cis-Pro content in intrinsically disordered proteins like alpha-synuclein is low and less sensitive to factors like ionic strength.
• These findings have implications for understanding protein folding mechanisms and enzyme recognition of proline conformers.