Examination of Protein Folding Using Sivvu™
Matthew Haveman and Prof. Douglas A. Vander Griend, Ph.D., Department of Chemistry and Biochemistry, Calvin College
Introduction
Method
Some proteins will assume partially folded, stable forms in
solution when in the presence of a denaturing agent. The
number of intermediate forms for a given protein can be
determined using Sivvu™ to analyze the UV-Vis absorbance
data from a titration.
• 30 solutions of BSA (Bovine Serum Albumin) were
prepared with concentrations of GdnHCL (Guanidine
Hydrochloride) ranging from 0 M to 7 M.
• Sivvu™ was used to determine the number of unique
forms of BSA in solution and fit the data to the
generalized equilibrium equation:
Sivvu™ uses equilibrium-restricted factor analysis to
determine the number of absorbing species in solution, the
molar absorptivity values, and the equilibrium constants.
P + n * GdnHClm
• m: number of GdnHCl molecules treated as a unit
• n: number of units of GdnHCl required to transform the
protein
Crystal Structure of Bovine Serum Albumin (BSA)
Molar Absorptivity
Concentration
P’
Residuals
Raw Absorbance
Results
Future Work
• Factor analysis suggested the presence of three forms
of BSA in solution:
• Folded
• Partially folded
• Unfolded
• Additional titrations with high-purity BSA under a
variety of denaturing conditions:
• Temperature
• pH
• Organic solvents
• Three forms of BSA require two equilibrium equations:
• Synthesize a chromophore for BSA to fold around.
PF + n1 * GdnHClm
P’ + n2 * GdnHClm
n1
15
n2
6300
m
100
P’
PU
R2
0.999172
• Determine the maximum number of forms Sivvu™
can distinguish using artificial datasets.
RMS Residual
0.00504783
Acknowledgments
Graph of factor significance when reconstructing data.
This work was supported by the Petroleum
Research Fund of the American Chemical Society.