Protein Stability and Folding

In addition to structural differences among protein isoforms, biophysical properties are important determinants of their functional properties. An emerging concept in protein folding is the stable folding intermediate, also referred to as the molten globule state. The molten globule is a semirigid structure that is almost as compact as the native structure. It retains most of the secondary structure of the native state and much of the tertiary structure; owing to the partial loss of tertiary structure, it usually contains an exposed hydrophobic surface. Until recently, it had been assumed that the molten globule was a relatively rare occurrence. However, there is a large body of experimental evidence that the molten globule state is a common feature of most proteins, can exist in cells, and plays a key role in a wide variety of physiological processes, including translocation across membranes, increased affinity for membranes, binding to liposomes and phospholipids, protein trafficking, extracellular secretion, and the control and regulation of the cell cycle.

To compare the physical characteristics of the apoE isoforms, we conducted guanidine, urea, and thermal denaturation studies of apoE2, apoE3, and apoE4 and their 22- and 10-kDa fragments. Guanidine and urea denaturation demonstrated that the two domains unfold independently in the three isoforms. However, all three denaturation methods showed differences in stability among the amino-terminal fragments of the apoE isoforms. ApoE4 denatured at the lowest concentrations of guanidine and urea and at the lowest temperature, while apoE2 denatured at the highest concentrations and temperature. Furthermore, guanidine and urea denaturation showed that apoE4, unlike apoE2 and apoE3, did not fit a two-state denaturation equilibrium. The lack of cooperative unfolding suggests that apoE4 forms a stable, partially folded intermediate.

Since unfolding intermediates are often more stable at an acidic pH, we examined urea denaturation of the three 22-kDa fragments at pH 4.0. The results demonstrated two-phase denaturation for apoE4 and a shoulder in the denaturation curve of apoE3, similar to that seen with apoE4 at pH 7.0. These findings suggest that both apoE3 and apoE4, but not apoE2, display folding intermediates at pH 4.0 in urea. Analysis of denaturation curves using a three-stage model revealed that the intermediate in apoE4 represents approximately 90% of the mixture at 3.75 M urea, whereas in apoE3 the intermediate represents approximately 30% of the total protein population at this urea concentration and can be increased to approximately 80% at 4.75 M urea.

In collaboration with Drs. Anthony Fink and Keith Oberg (University of California, Santa Cruz), we examined the apoE4 folding intermediate in the absence and presence of urea at pH 4.0 with their novel method of Fourier transmittance reflective infrared analysis. In the absence of urea, the secondary structure of the apoE4 22-kDa fragment was estimated to consist of 75% a-helix and 3% b-sheet, consistent with previous estimates. In 3.75 M urea, apoE4 consisted of 46% a-helix and 17% b-sheet. Thus, the apoE4 intermediate contained 61% of the original helical content and had an increased b-sheet structure. Pepsin proteolysis of apoE4 at pH 4.0 in 0 M and 3.75 M urea showed cleavages between helices 2 and 3, within helix 3, and between helices 3 and 4 in the presence of urea. These results indicate that there is a conformational change in apoE4 at pH 4.0 in the presence of urea. We speculate that the four-helix bundle is partially unfolded, similar to the unfolded structure when this fragment binds to lipid.

The results from the characterization of the apoE4 stable folding intermediate indicate that this intermediate is a molten globule. Since molten globules have been implicated in a variety of physiological processes, including membrane binding and translocation, we examined the ability of apoE4 and apoE3 to bind and disrupt DMPC vesicles at pH 4.0, with and without urea. Under both conditions, apoE4 was more effective than apoE3, suggesting that the apoE4 molten globule may be involved in membrane translocation.

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