Keeping it in the family: folding studies of related proteins.

Investigators have recently turned to studies of protein families to shed light on the mechanism of protein folding. In small proteins for which detailed analysis has been performed, recent studies show that transition-state structure is generally conserved. The number and structures of populated folding intermediates have been found to vary in homologous families of larger (greater than 100-residue) proteins, reflecting a balance of local and global interactions.

Multiple roles of prolyl residues in structure and folding.

To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I). While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics. The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process. By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding. Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly. Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble. Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state.

Unfolding dynamics of a beta-sheet protein studied by mass spectrometry.

The unfolding dynamics of cellular retinoic acid-binding protein I (CRABP I), an 18 kDa predominantly beta-sheet protein, were studied by monitoring the hydrogen-deuterium (H-D) exchange reaction under various solution conditions. A bimodal charge state distribution was observed when a denaturing agent was added to the protein aqueous solution. These two populations exhibit different kinetics of H-D exchange, with the high charge state ions undergoing very rapid isotope exchange, while the low charge state protein ions exchange cooperatively but at much slower rates. Transiently populated intermediate states were detected indirectly using hydrogen exchange measurement in aqueous solution at various pHs. At pH 2.5 and room temperature, three distinct populations of CRABP I ions exist over an extended period of time, each corresponding to a specific degree of backbone amide hydrogen atom protection. Mass spectral data are complementary to hydrogen exchange measurements by NMR, since the former samples a much faster time-scale of dynamic events in solution.

Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry.

The conformation of a three-disulphide derivative of bovine alpha-lactalbumin bound to the molecular chaperone GroEL has been investigated by monitoring directly its hydrogen exchange kinetics using electrospray ionization mass spectrometry. The bound protein is weakly protected from exchange to an extent closely similar to that of an uncomplexed molten globule state of the three-disulphide protein. Binding to GroEL in this system appears to involve relatively disordered partly folded states resembling intermediates formed in the very early stages of kinetic folding of many proteins in vitro.

Kinetic consequences of the removal of a disulfide bridge on the folding of hen lysozyme.

Quenched-flow hydrogen exchange labeling, monitored by 1H NMR and electrospray ionization mass spectrometry (ESI-MS), has been employed in conjunction with stopped-flow circular dichroism and fluorescence to study the kinetic refolding from guanidinium chloride of a derivative of hen lysozyme in which one of the four disulfide linkages (Cys6-Cys127) has been selectively chemically reduced and carboxymethylated (CM6,127-lysozyme). Removal of this disulfide bridge has little effect on the structure and activity of the native enzyme, and the overall kinetics of refolding are very similar to those of the unmodified protein. A substantial amount of secondary structure is formed within 2 ms of the initiation of folding, followed by the slower formation of tertiary interactions characteristic of the native state, which are attained with a time constant (tau) of ca. 200 ms. There is clear evidence for fast and slow refolding populations, as in the intact protein. Folding of the three-disulfide derivative does, however, exhibit a major difference from that of the intact protein under the same final refolding conditions, in that the transient intermediate on the major refolding pathway of the intact protein, having persistent structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labeling during folding of the three-disulfide derivative. This suggests that the disulfide bond linking the N- and C-terminal regions of the protein is crucial for stabilization of the partially folded intermediate. In addition, the overshoot in the far-UV CD and the fluorescence minimum, both of which are attributed to non-native interactions, is not observed in the folding of CM6,127-lysozyme. That the lack of a detectable stable intermediate in the folding of CM6,127-lysozyme does not significantly affect the rate of attainment of the native state of the protein supports the proposed independent nature of the two folding domains and, as the Cys6-Cys127 disulfide bond is located in the alpha-domain, indicates that the rate-limiting step in folding of the intact protein, as well as of the three-disulfide derivative, involves stabilization of the beta-domain. The role of disulfide bridges in the formation and maintenance of the three-dimensional fold of proteins and in facilitating the observation of marginally stable intermediate species is discussed.

Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme.

Differential scanning calorimetry experiments as a function of pH have been carried out for native hen egg white lysozyme and a three-disulphide derivative (CM6,127-lysozyme). The results indicate that the enthalpy (delta H298) and heat capacity changes (delta Cp) for unfolding are closely similar for the two proteins. This shows that the substantial reduction (25 degrees C at pH 3.8) in Tm resulting from removal of the 6-127 disulphide bond can, to a good approximation, be attributed totally to an increase in the entropy difference between the native and denatured states. The significance of this result for understanding the factors influencing the stability of folded proteins is discussed.