Folding and signaling share the same pathway in a photoreceptor

Abstract

The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.

Figure 1

Protein folding in PYP. (A) The kinetics of refolding in PYP observed by stopped-flow absorbance spectroscopy. Refolding was initiated by a jump in denaturant concentration, obtained by rapid mixing, from 3.5 to 1.0 M GdmHCl at pH 7.0. The resulting kinetics were monitored at 340 (dashed lines) and 445 (solid lines) nm for the refolding of pU^trans (monoexponential decay with k = 31.4 s⁻¹) and a mixture of pU^trans and pU^cis generated by the illumination of pU^trans with near-UV light [biexponential decay with k₁ = 31.4 s⁻¹ (68%) and k₂ = 0.9 s⁻¹ (32%)], The kinetics of the pB^cis to pG^trans photocycle transition (○; monoexponential decay with k = 0.9 s⁻¹) were determined under identical conditions, and normalized to match the amplitude of the rapid mixing signals. (B) Chevron analysis of protein folding in PYP. The kinetics of pG^trans unfolding (●) and of pU^trans (●) and pU^cis (▴) refolding were determined as a function of GdmHCl concentration. The kinetics of the pB^cis to pG^trans photocycle kinetics (○) were determined under identical conditions. The rapid mixing data were fit (solid lines, see Tables 1 and 2) to a two-state model for PYP containing a trans chromophore, and to a model containing an on-pathway intermediate for PYP containing a cis chromophore. (C) Equilibrium denaturation titration of pG^trans to pU^trans with GdmHCl, followed by visible absorbance spectroscopy at 340 nm (○) and 446 nm (▵).

Figure 2

Specific denaturation of the pB^cis photocycle intermediate. (A) Equilibrium denaturant titrations of pG^trans (squares) and pB^cis (circles) with GdmHCl (closed symbols) and urea (open symbols). The pG^trans denaturation curve was determined from absorbance data at 340 and 446 nm; pB^cis denaturation was monitored at 375 nm in the pB^cis-pG^trans absorbance difference spectrum. (B) CD spectroscopy of the pU^cis state (●) was performed by probing the CD signal during pG^trans recovery after PYP photoexcitation at various wavelengths. The depicted values were obtained by extrapolation to t = 0 (i.e., immediately after closure of the optical shutter for sample illumination) for pU^cis (●) and t = ∞ (i.e., after complete pG^trans recovery) for pG^trans (○). The pU^cis values were scaled to 100% pG^trans photobleaching, as determined from the measured absorbance values at 446 nm. For comparison, the steady-state CD spectrum of the pG^trans state in the presence of 0 (solid line) and 6.0 (dotted line) M urea are shown. (C) The monoexponential recovery of pG^trans in the presence of 6.0 M urea, detected by CD spectroscopy at 222 nm. (D) The amplitude of the light-induced loss of CD signal at 222 nm extrapolated to t = 0 (●) was determined as a function of urea concentration and scaled to 100% pG^trans photobleaching. ○ depict the dependence of the CD signal for pG^trans at 222 nm.

Figure 3

Integrating folding and signaling in PYP. The folding and photocycle transitions in PYP are depicted schematically. Note that pU^cis is an off-pathway intermediate for the photocycle, whereas pB^cis is an on-pathway intermediate for pU^cis refolding.