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. 2009 Jun;75(4):799-806.
doi: 10.1002/prot.22286.

Comparison of the folding mechanism of highly homologous proteins in the lipid-binding protein family

Ira J Ropson  1 , Joshua A Boyer, Blake A Schaeffer, Paula M Dalessio
Affiliations

Comparison of the folding mechanism of highly homologous proteins in the lipid-binding protein family

Ira J Ropson et al. Proteins. 2009 Jun.

Abstract

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.

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Figures

Figure 1
Figure 1
Cartoon structure of hBABP (pdb code 1o1u).
Figure 2
Figure 2
Fluorescence spectra of native hBABP (○), rBABP (×), 6FTrp-hBABP (□), and 6FTrp-rBABP (◊) in buffer, and unfolded 6FTrp-rBABP (●) in buffer with 8.0 M urea.
Figure 3
Figure 3
Dependence of fluorescence (370 nm,□) and CD (218 nm, ○) intensity on urea concentration for hBABP.
Figure 4
Figure 4
Dependence of the rates of folding and unfolding on urea concentration by fluorescence (open symbols) and CD (closed symbols) for hBABP. The gray lines are the folding and unfolding rates for rBABP fit to an exponential dependence on denaturant concentration.
Figure 5
Figure 5
Spectra of 6FTrp-rBABP at the listed concentrations of urea. The chemical shift scale is from 3-fluoro-tyrosine.
Figure 6
Figure 6
6A. Dependence of fluorescence intensity on urea concentration for 6FTrp-rBABP (□). 6B. Dependence of fluorescence intensity (□) and NMR population (●) on urea concentration for 6FTrp-hBABP. The line through the data is that of a fit to a two state model for stability for each data set. The gray lines in each figure are the fit to the unlabeled protein.
Figure 7
Figure 7
Spectra of 6FTrp-hBABP at the listed concentrations of urea. The chemical shift scale is from 3-fluoro-tyrosine.
Figure 8
Figure 8
Dependence of the rates of folding and unfolding on urea concentration for WT-rBABP (open symbols) and 6Ftrp-rBABP (closed symbols). 3B. Dependence of the rates of folding and unfolding on urea concentration for WT-hBABP (open symbols) and 6FTrp-hBABP (closed symbols).
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References

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