Folding of intracellular retinol and retinoic acid binding proteins.
The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.
Burns
L L
LL
Department of Biochemistry and Molecular Biology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, 17033 USA.
Ropson
I J
IJ
D000595:Amino Acid Sequence; D000818:Animals; D007424:Intracellular Fluid; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000737:chemistry*; D012177:Retinol-Binding Proteins / Q000737:chemistry*; D054840:Retinol-Binding Proteins, Cellular
C518460:Rbp2 protein, mouse; C518461:Rbp2 protein, rat; D018168:Receptors, Retinoic Acid; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular