Sequence composition effects on denatured state loop formation in iso-1-cytochrome c variants: polyalanine versus
Franco O Tzul1, Eydiejo Kurchan, Bruce E Bowler
1Department of Chemistry and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA.
Protein loop formation, crucial for folding, is not significantly aided by glycine's flexibility compared to alanine. Steric properties, not just flexibility, influence loop stability in proteins like cytochrome c.
Area of Science:
- Biochemistry
- Protein Folding Dynamics
- Molecular Biophysics
Background:
- Protein folding initiates with the formation and stability of early loops.
- Loop formation and persistence are influenced by the steric properties of amino acid residues.
Purpose of the Study:
- Investigate the role of residue steric properties in protein loop formation and stability.
- Examine how substituting alanine with glycine affects the denatured state and loop stability of iso-1-cytochrome c.
Main Methods:
- Utilized guanidine hydrochloride (Guanidine-HCl) unfolding experiments to assess protein stability.
- Introduced alanine inserts and progressively substituted them with glycine in iso-1-cytochrome c.
- Measured histidine-heme loop stability in 3 M Guanidine-HCl across protein variants.
Main Results:
- All protein variants exhibited similar free energies of unfolding (approx. 2 kcal/mol).
- Histidine-heme loop formation stabilized the denatured state in all variants.
- Glycine substitution for alanine had a minimal impact on equilibrium loop stability.
Conclusions:
- The steric properties of residues, rather than main-chain flexibility, are key determinants of early loop stability.
- Increased main-chain flexibility from glycine does not enhance the persistence of simple loops during protein folding.
- Polyalanine and polyglycine inserts show comparable loop behavior due to underlying structural and kinetic factors.
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