Electronic Supplementary Material

For

Folding dynamics of phenylalanine hydroxylase depends on the enzyme’s metallation state: the native metal, iron, protects against aggregate intermediates

Aristobulo Loaiza,¶ Judith A. Ronau,¶ Alexander Ribbe,¶ Lia Stanciu,¥ John. W. Burgner II,§ Lake N. Paul,§* and Mahdi M. Abu-Omar¶*

¶ Brown Laboratory, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA

¥ School of Materials Engineering, Purdue University, Neil Armstrong Hall of Engineering, 701 West Stadium Avenue, West Lafayette, IN 47907, USA

§ Bindley Bioscience Center, Discovery Park, Purdue University, 1203 W. State Street, West Lafayette, IN 47907, USA

* To whom correspondence should be addressed (; )


Figure S1 CD spectra of apo-cPAH at different concentrations of GuHCl

Figure S2 CD spectra of Co-cPAH at different concentrations of GuHCl

Figure S3 Comparison of CD spectra for apo- versus holo- (Co) cPAH in 2.6 M GuHCl

Figure S4 Unfolding (squares) and refolding (inverted triangles) profiles for apo-cPAH

Figure S5 Refolding CD spectra for Co-cPAH. Dashed spectrum: 30 mM Co-cPAH in 3.5 M GuHCl. Solid spectrum: refolded Co-cPAH upon 4-fold dilution to give 7.5 mM Co-cPAH in 0.875 M GuHCl.

Figure S6 Refolding CD spectra for apo-cPAH. Dashed spectrum: 30 mM apo-cPAH in 3.5 M GuHCl. Solid spectrum: refolded apo-cPAH upon 4-fold dilution to give 7.5 mM apo-cPAH in 0.875 M GuHCl.

Figure S7 GuHCl dependence on ANS emission intensity (open circles) alongside the CD data (hatched squares) for apo-cPAH

Figure S8 ANS emission intensity for holo-cPAH

Emission intensity is plotted as a function of guanidine HCl after incubation with Co-cPAH. Inset shows ANS emission spectra in the presence of Co-cPAH at different concentrations of denaturant GuHCl (0, 1.0 and 2.2 M).

Figure S9 GuHCl dependence on ANS emission intensity (filled triangles) alongside the CD data (open squares) for native Fe-cPAH

Figure S10 TEM (Transmission Electron Microscopy) micrograph of apo-cPAHI in 2.6 M GuHCl, showing aggregate protein formation of 10+ nm in size

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