DETERMINATION OF MEMBRANE PROTEIN STABILITY VIA
THERMODYNAMIC COUPLING OF FOLDING TO
THIOL-DISULFIDE INTERCHANGE
Electronic Supplementary Material
Lidia Cristian, James D. Lear*, William F. DeGrado*
Department of Biochemistry & Biophysics, University of Pennsylvania School of
Medicine, Philadelphia PA, 19104-6059 U.S.A.
Running title: Determination of Membrane Protein Stability
*
Authors to whom correspondences should be addressed.
Addresses: Department of Biochemistry & Biophysics
University of Pennsylvania School of Medicine
1010 Stellar-Chance Building
Philadelphia PA, 19104-6059 U.S.A.
Phone: (215) 898-4590
Fax: (215) 573-7229
Email: wdegrado@mail.med.upenn.edu
The electronic supplementary material consists of 2 pages describing the procedure
utilized to fit the data and the legends for two supplementary figures. File prepared using
Microsoft Word 2000 from a PC.
1
The model presented in Scheme 2 was utilized to derive the equation for fitting the data
in Figure 5. For fitting model parameters to the experimental data, the equilibrium
concentrations of all species present were expressed as a function of known peptide and
detergent concentrations using the equilibria in Scheme 2. The material balance in
Scheme 2 can be expressed as:
Equation 1
MSH + 4 TSH, SH + 4 T SS,SH + 4 T SS,SS + 2 DSS = Pt
where Pt is the total protein concentration.
MSH, TSH,
SH,
TSS,SH, TSS,SS and DSS concentrations, calculated as functions of MSH from the
corresponding equilibrium constants defined in the text, were substituted in Equation 1 to give:
Equation 2
MSH + 4MSH4/K1+4xMSH4/(K1K2)+4x2MSH4/(K1K2K3)+2[MSH4x2K4/(K1K2K3)]0.5–Pt = 0
where x represents [GSSG]/[GSH]2. This equation, solved using iterative root-finding
algorithms in Igor Pro was used to individually fit data sets at fixed x in Figure 5. All
parameters were allowed to freely vary. As initial guesses for the curve fit, we used K 1
and K4 values obtained from the equilibrium sedimentation study. K2 and K3 were given
close starting values; generally K3 was chosen ~ (1/3) K2, as discussed in the text. The
parameters fitted to the data are shown in Table 1. In fitting the data, we found it
necessary to include in the fitting function a baseline correction factor, g. This parameter
was also allowed to vary in all fits. This parameter presumably reflects the presence of
some fraction of the peptide existing as disulfide-bonded species, which is insensitive to
the redox potential of solution (probably due to the tendency of the peptide to form non-
2
specific aggregates). The values obtained for g were: 20.6, 42.1 and 39.2 corresponding
to the data sets for 0.1, 0.2 and 0.5 GSSG/GSH ratios, respectively (see Figure 5).
3
Supplementary Figures
Figure S1. Equilibrium analytical ultracentrifugation results for reduced (a) and oxidized
(b) peptide in 15 mM DPC at 25 0C. The peptide-to-DPC ratio was 1:150 in both cases.
The lines are best fits to a monomer-tetramer equilibrium model (a) and dimer-tetramer
association scheme (b). The residuals of the fits are shown in the upper panels.
4
Figure S2. Plot showing the reversibility of the disulfide-bonding in DPC micelles. The
circles represent the data when the equilibration is carried out starting with the reduced
peptide. Diamonds correspond to data obtained in the reverse measurements when the
equilibrium is approached from the other direction by starting with the dimer. The ratio
of peptide/detergent was 1:50 in both experiments.
5