Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein.
The equilibrium and kinetic properties of the unfolding-refolding transitions of Escherichia coli derived rat intestinal fatty acid binding protein have been examined using several different denaturants. This protein, which contains 2 tryptophans but no prolines or cysteines, has a predominantly beta-structure: its 10 antiparallel beta-strands are organized into 2 orthogonal sheets surrounding a large solvent-filled internal cavity. For urea and guanidine hydrochloride, the completely reversible transition was monitored by circular dichroism, absorbance, and fluorescence spectroscopy. Each of these data sets was best fit by a simple, two-state model involving only native and unfolded forms. However, linear extrapolation to determine the free energy of folding in the absence of denaturant resulted in different values for the free energy of folding depending upon which denaturant was used. When fluorescence was used to monitor the transition, the extrapolated free energy estimates for the two denaturants were markedly different: 10.03 +/- 0.24 kcal mol-1 for urea versus 5.22 +/- 0.33 kcal mol-1 for guanidine hydrochloride. The midpoints of these transitions were 5.51 and 1.36 M, respectively. The transition caused by either denaturant as monitored by circular dichroism and absorbance spectroscopy was virtually coincident with that monitored by fluorescence, further supporting the assignment of a two-state model for the equilibrium results. The addition of a 2-fold molar excess of ligand (oleate) increased the extrapolated estimates approximately 2.5 kcal mol-1 for both denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)
Ropson
I J
IJ
Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, Missouri 63110.
Gordon
J I
JI
Frieden
C
C
D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012984:Software; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001059:Apoproteins; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D006146:Guanidines; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019791:Guanidine