Hydrogen/Detureium Exchange Rate
Folding rate determination and peptide binding complex

 

Result

Native levels of both intrinsic fluorescence (97 ( 3%)(A) and peptide binding activity (95 ( 6%) (B) are recovered with effectively identical kinetics (25.6 +/-1.2 and 23.7 +/- 2.0 s -1 , respectively, in 0.727 M GuHCl at 20 ¢XC). Consistent with this observation, the native NMR signal intensity (95 ( 8%) is recovered within the 2 s dead time of kinetic NMR measurements (C), indicating that a native core geometry is recovered within that time frame. The expected fluorescence of the unfolded protein in the refolding buffer (40%) is indicated in (A) by an arrow. It was calculated by linear extrapolation of the fluorescence of denatured Fyn-SH3 at high GuHCl concentrations. (Figure adopted from Plaxco, et. al., Biochemistry, 1998, 37(8):2529)
Diagrammatic representation of the Fyn-SH3-peptide complex. The peptide is shown in ball-and-stick representation, while the protein is shown as a backbone worm. The structure of Pro134 is clearly constrained by Trp119 and Tyr137, and this observation, together with molecular dynamics simulations ,strongly suggests that a cis-proline cannot be accommodated at this position without significantly disrupting both the peptide binding surface and the intrinsic fluorescence of the protein.

 

Kinetic determination of folding rate
(A) Refolding kinetics of src SH3 in 0.4 M GdmCl at 295 K followed by stopped-flow fluorescence. The slow phase due to proline isomerization accounts for ~10% of the total change in signal. The baseline for the denatured protein in 0.4 M GdmCl is assumed to be the same as that in 4 M GdmCl since equilibrium GdmCl denaturation did not reveal any dependence of the fluo-rescence signal of the unfolded protein on GdmCl. (B) GdmCl dependence of ln kobs at 295 K. Closed circles are averages of the rates obtained from 5-7 folding or unfolding experiments at a particular GdmCl concentration. (Figure adopted from Grantcharova and Baker, Biochemistry, 1997, 36(50):15685)
GU = Hg + mD - TSg Cp [Tg - T + T ln(T/Tg)]
Hg and Sg are the enthalpy and entropy changes upon unfolding at Tg. Cp, the change in heat capacity, and m, the dependence of GU on [GdmCl], are both assumed to be independent of temperature and [GdmCl]. D is the GdmCl concentration in (molar), Tg is the reference tem-perature (295 K), and T is the absolute temperature. A second fit to the data included individual dependences of H, S, and Cp on GdmCl concentration.
Figure adopted from Grantcharova and Baker, Biochemistry, 1997, 36(50):15685
The rates in 0 M GdmCl and the kinetic m values were obtained by fitting kinetic data to:
ln kobs = ln [kfH20exp(-mf[GdmCl]/RT) + kuH2Oexp(m u[GdmCl]/RT)]
a slow phase (k = 0.017 +/- 0.002 s-1) due to proline isomerization was observed.

 

Folding rate comparison
Src-SH3a
Fyn-SH3b
Alpha-spectrinc
kf
ku
kf
ku
kf
ku
56.7
0.007
94.3
0.001
2.83
0.1

a. Grantcharova and Baker, Biochemistry, 1997, 36(50):15685

b. Plaxco, et. al., Biochemistry, 1998, 37(8):2529

c. Viguera, et al., J. Mol. Biol., 1995, 247:670

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Danny S. Hsu 1999-2000. All rights reserved.