Comparison of the folding mechanism of highly homologous proteins in the lipid-binding protein family.
The folding mechanism of two closely related proteins in the intracellular lipid-binding protein family, human bile acid-binding protein (hBABP), and rat bile acid-binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two-state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6-fluorotryptophan-labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding.
Ropson
Ira J
IJ
Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania, USA. iropson@psu.edu
Boyer
Joshua A
JA
Schaeffer
Blake A
BA
Dalessio
Paula M
PM
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D014508:Urea / Q000737:chemistry; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014364:Tryptophan; D014443:Tyrosine; D014508:Urea; C037643:bile acid binding proteins; C001245:3-fluorotyrosine; C001244:6-fluorotryptophan; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)