Abstract
Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.
Highlights
We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid chromophore
Summary—We report a novel strategy to examine residual structure in the fully unfolded state based on comparing the unfolding transitions of the native state with those of partially unfolded states of the same protein
The results indicate that residual structure in the fully unfolded state of the blue-light receptor PYP is significantly modified by the isomerization of its lightsensitive p-coumaric acid (pCA) chromophore
Summary
Protein Purification and Spectroscopy—PYP was overexpressed in Escherichia coli and purified as described previously (29). The pBdark state was studied at pH 2.0 in a buffer of 10 mM potassium phosphate, 25 mM citrate, and 20 mM HCl. Thermal Denaturation—Equilibrium thermal denaturation curves for pG were measured using a Cary300 UV-visible spectrophotometer (Varian) with a thermostated cuvette holder flushed with nitrogen gas. Thermal denaturation of the pB photocycle intermediate was studied by measuring steady state light-induced difference spectra with a Hewlett-Packard 8453 diode array spectrophotometer at a range of temperatures. Equilibrium Denaturant Titrations—Equilibrium GdmHCl titrations of PYP with absorbance detection were performed at pH 2.0 in 10 mM potassium phosphate (pH 7.3), 25 mM citrate, and 20 mM HCl at 25 °C using a Cary 300 UV-visible spectrophotometer (Varian) with a thermostated cuvette holder. The ⌬Cp for the unfolding of the pBdark state was obtained by measuring GdmHCl titration curves at various temperatures in the range 4 –35 °C. Perature dependence of ⌬Gu(H2O) was analyzed using Equation 1 to extract the ⌬Cp for pBdark unfolding
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