UV-resonance raman thermal unfolding study of Trp-cage shows that it is not a simple two-state miniprotein.
Trp-cage, a synthetic 20 residue polypeptide, is proposed to be an ultrafast folding synthetic miniprotein which utilizes tertiary contacts to define its native conformation. We utilized UV resonance Raman spectroscopy (UVRS) with 204 and 229 nm excitation to follow its thermal melting. Our results indicate that Trp-cage melting is complex, and it is not a simple two-state process. Using 204 nm excitation we probe the peptide secondary structure and find the Trp-cage's alpha-helix shows a broad melting curve where on average four alpha-helical amide bonds melt upon a temperature increase from 4 to 70 degrees C. Using 229 nm excitation we probe the environment of the Trp side chain and find that its immediate environment becomes more compact as the temperature is increased from 4 to 20 degrees C; however, further temperature increases lead to exposure of the Trp to water. The chi(2) angle of the Trp side chain remains invariant throughout the entire temperature range. Previous kinetic results indicated a single-exponential decay in the 4-70 degrees C temperature range, suggesting that Trp-cage behaves as a two-state folder. However, this miniprotein does not show clear two-state behavior in our steady-state studies. Rather it shows a continuous distribution of steady-state spectral parameters. Only the alpha-helix melting curve even hints of a cooperative transition. Possibly, the previous kinetic results monitor only a small region of the Trp-cage which locally appears two-state. This would then argue for spatially decoupled folding even for this small peptide.
Ahmed
Zeeshan
Z
Department of Chemistry, University of Pittsburgh, PA 15260, USA.
Beta
Ilir A
IA
Mikhonin
Aleksandr V
AV
Asher
Sanford A
SA
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014466:Ultraviolet Rays
D010455:Peptides; C508725:Trp-cage peptide; D011392:Proline