A specific hydrophobic core in the α-lactalbumin molten globule¹

Abstract

Molten globules are partially structured protein folding intermediates that adopt a native-like overall backbone topology in the absence of extensive detectable tertiary interactions. It is important to determine the extent of specific tertiary structure present in molten globules and to understand the role of specific side-chain packing in stabilizing and specifying molten-globule structure. Previous studies indicate that a small degree of specific side-chain packing stabilizes the structures of the cytochrome c, apomyoglobin, and staphylococcal nuclease molten globules. Here we investigate the extent of specific side-chain packing in the molten globule of α-lactalbumin (α-LA), a highly fluctuating, non-cooperatively formed molten globule. By analyzing a set of point mutations in the helical domain of α-LA, we have identified a stabilizing hydrophobic core. Moreover, this core corresponds to a previously identified structural subdomain and likely contains some native-like packing interactions. Our results suggest that native-like packing of core amino acids helps stabilize molten globules and that some specific interactions can exist in even highly dynamic, fluctuating species.

Introduction

Molten globules are compact, partially structured forms of proteins thought to be general intermediates in protein folding Dobson 1992, Kuwajima 1989, Ptitsyn 1992. Molten globules have high levels of native-like secondary structure arranged in an overall native-like fold (Peng et al., 1995a), but lack rigid side-chain packing and extensive detectable tertiary interactions. As such, molten globules can be viewed as a “low-resolution solution” to the protein folding problem, in which a native-like protein architecture has been established. It is important to understand how an overall native-like fold can be formed in the apparent absence of extensive specific tertiary interactions. One extreme possibility is that molten globule structure is formed by non-specific hydrophobic collapse around a core of dynamic, loosely interacting residues. In this case, specific packing interactions may be minimal or non-existent, and molten globule structure may be determined by the global distribution of hydrophobic and hydrophilic amino acids, as opposed to specific amino acid identity. Another extreme possibility is that undetected, highly specific tertiary interactions stabilize molten globules. In this case, specific interactions amongst a small subset of key residues may be obscured by high conformational mobility in the remainder of the molecule.

The extent of specific native packing that exists in the molten globules of apomyoglobin, cytochrome c, and staphylococcal nuclease has been assessed by studying point mutants of hydrophobic core residues Carra et al 1994, Colon and Roder 1996, Colon et al 1996, Kay and Baldwin 1996, Lin et al 1994, Marmorino and Pielak 1995. In these studies, the effects of mutations on the stabilities of the molten globule states are small, suggesting that tertiary interactions are only partially formed in the molten globule. Nonetheless, the effects of point mutations on the stabilities of the native and molten globule states are correlated. These results suggest that a small degree of specific native-like packing stabilizes and helps determine the structure of the molten globule.

The molten globule folding intermediate of α-lactalbumin (α-LA), a two domain protein containing four disulfide bonds, consists of a native-like helical domain and a largely unstructured β-sheet domain Alexandrescu et al 1993, Kuwajima 1996, Schulman et al 1995, Wu et al 1995. Molten globules can range in orderliness from highly dynamic species with poor NMR chemical shift dispersion and non-cooperatively formed structure, to highly ordered species with substantial NMR chemical shift dispersion and cooperatively formed structure Alexandrescu et al 1993, Feng et al 1994, Redfield et al 1994. The α-LA molten globule is highly dynamic, yielding NMR spectra with broad linewidths and poor chemical shift dispersion. Moreover, formation of the α-LA molten globule is largely non-cooperative, as judged by proline scanning mutagenesis and denaturation transitions monitored at global and residue-specific levels Schulman and Kim 1996, Schulman et al 1997, Shimizu et al 1993. Furthermore, thermal denaturation of the α-LA molten globule is accompanied by little excess heat absorption, suggesting that the core of the molten globule may be loosely ordered and solvent exposed, although this is in debate Pfeil et al 1986, Xie et al 1991, Yutani et al 1992. The extent of specific packing interactions in such a dynamic fluctuating species is unclear, but may be elucidated by systematic mutagenesis in the core of the molten globule.

There are two hydrophobic cores in the structure of native α-LA (Figure 1; Acharya et al., 1991). One, called the hydrophobic box, comprises residues from the C and D-helices and the β-sheet domain (Acharya et al., 1991). Another comprises residues from the A, B, and 3₁₀-helices. Measurements of the equilibrium constants for formation of native and non-native disulfide bonds in the helical domain of the α-LA molten globule indicate that the region around the 28–111 disulfide bond plays an important stabilizing role (Peng et al., 1995b). Although the 28–111 disulfide bond connects the B and D-helices, which lie near the hydrophobic box, no direct evidence indicates that the hydrophobic box forms the stabilizing core of the molten globule. On the other hand, NMR studies delineate a stable structural subdomain in the α-LA molten globule, comprising the A, B, and 3₁₀-helices (Schulman et al., 1997).

Here we analyze a collection of point mutants in the helical domain of α-LA, using the equilibrium constant for formation of the 28–111 disulfide bond to monitor effects on the stability of the molten globule. We find that residues from the A/B/3₁₀ subdomain, as opposed to the hydrophobic box, form the major stabilizing hydrophobic core. Moreover, this core likely contains some specific native-like packing interactions that may help specify the native-like structure of the α-LA molten globule.