Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Abstract

Photochemically induced dynamic nuclear polarization NMR pulselabeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human ␣-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the ␣-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and nonnative environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.hydrophobic cluster ͉ protein folding ͉ pulse labeling ͉ chemically induced dynamic nuclear polarization ͉ molten globule V ery significant advances, both theoretical and experimental, have been made in recent years in elucidating the principles that govern the folding of proteins (1, 2). In particular, the concept of an energy landscape has provided a general framework for interpreting the thermodynamics and kinetics of the folding process through which a polypeptide chain converts from a disorganized unfolded state into the tightly packed native state (N state) at the global energy minimum (3, 4). Recent approaches, involving a combination of computer-simulation techniques with experimental constraints, have enabled three-dimensional structural ensembles of various partially folded species and folding intermediates to be defined and provide a coarse-grained description of the development of interresidue interactions and chain topologies involved in attaining the final native structure (5-7). Of particular importance in this context are compact denatured states, often known as molten globules (MGs), which are commonly characterized by the presence of native-like secondary structure and hydrodynamic radii but lack both native-like packing of the internal amino acid side chains and exclusion of solvent molecules from the hydrophobic core of the protein (8, 9). The detailed experimental description of such states will undoubtedly provide important information about the specific factors stabilizing the overall chain topology at a residue-specific side-chain level (10). However, MG states are very difficult to characterize by using conventional x-ray crystallography or NMR techniques because of their heterogeneous character (11,12).We have recently developed NMR techniques to characterize aromatic side chains in transient, kinetic intermediate species present in real-time protein folding experiments (13, 14) or in p...

Measurement of energy landscape roughness of folded and unfolded proteins

“…Population of this molten globule state leads to NMR line broadening, which indicates that the component species have trapping times on the millisecond timescale (46). Indeed, the NMR line broadening (47) and complex interconversion behavior over a wide range of timescales (48) that are typical features of molten globule states are readily accounted for by subdiffusional behavior on a rugged energy landscape, as proposed here.…”

Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP

“…Molten globules are believed to have dynamic hydrophobic cores due to the lack of signal in the near-UV CD spectrum and the broadening of NMR signals of aromatic groups (6,7). Recent studies have shown that the side chains in the α-lactalbumin molten globule exchange between several well defined conformations, representing a dynamic ensemble of states with specific tertiary interactions (9).…”

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

“…Under destabilizing conditions such as low pH, addition of cosolvents, high temperature, or combinations thereof, apo-␣-lactalbumin exists in an MG state. The MG state is characterized by the absence of long-lived tertiary structure, but it still contains a high degree of secondary structure (20,21), with a radius of gyration only Ϸ10% larger than the native state (22,23). The MG state of ␣-lactalbumin has recently attracted considerable interest when it was realized that it may act as an important component causing apoptosis of tumor cells (24).…”