Sequential Equilibrium Unfolding of a Four-helix Bundle Peptide Shows that Urea and Guanidine have Different Effects on Tertiary and Secondary Structure. A CD and Time-resolved Fluorescence Spectroscopy Study
(English)In: Biophysical Journal, ISSN 0006-3495Article in journal (Refereed) Submitted
In this paper we show, using time-resolved fluorescence and CD spectroscopy, that equilibrium unfolding of the homodimeric four-helix bundle peptide (KE2D15)2 occurs via different pathways depending on the denaturant. The initial effect of guanidine hydrochloride (GdHCl) and urea is similar insofar that the tertiary structure is slightly destabilized. This is accompanied by a slight increase in helicity, which we believe is due to stabilization of the backbone at the expense of hydrophobic core stability. With GdHCl we observe an almost immediate (≥2 M GdHCl) dissociation of the dimer into helical monomers, while the effect of urea is to stabilize the helices and induce a solvent- or urea-separated state that persists up to about 5 M urea. At high urea concentrations, the peptide exists in monomeric but helical form. The partial and full dissociation of the dimeric four-helix bundle is monitored through time-resolved fluorescence spectroscopy. Through the use of time-resolved fluorescence, we can assess the heterogeneity of the partly and fully denatured states even though the denaturation is carried out at equilibrium conditions. The width of the fluorescence lifetime distributions are analyzed in terms of conformational space of the peptide.
conformational dynamics, protein folding, chemical denaturants, time-resolved fluorescence spectroscopy, CD spectroscopy
IdentifiersURN: urn:nbn:se:uu:diva-109375OAI: oai:DiVA.org:uu-109375DiVA: diva2:272260