Characterization of a trifluoroethanol-induced partially folded state of alpha-lactalbumin.
The protein alpha-lactalbumin exists in a partially folded molten globule state at pH 2.0, the A state. This state is believed to be compact, possessing a similar amount of secondary structure to the native state but having a flexible tertiary structure comprised mainly of non-specific hydrophobic clustering of residues. Addition of trifluoroethanol (TFE) to bovine, human and guinea pig alpha-lactalbumin at pH 2.0 has been found in each case to induce a conformational transition in the A state as monitored by circular dichroism, nuclear magnetic resonance chemical shifts, and 1-anilinonaphthalene-8-sulphonate binding. The mid-point of this transition is near 15% (v/v) TFE and is effectively complete by 50% (v/v) TFE at 315 K. Far ultraviolet circular dichroism ellipticities at 208 nm and 220 nm, usually taken as a measure of the degree of helical character, are substantially more negative in the TFE state than in the A state. Furthermore, backbone amide protons protected from solvent exchange in the A state are generally at least as strongly protected in the TFE state; patterns of protection appear similar in the two states and include at least part of both the B and C alpha-helices. One major difference from the A state is nevertheless evident: the ability to bind the fluorescent probe 1-anilinonaphthalene-8-sulphonate, characteristic of molten globule states, is lost in the TFE state. Like the A state, the TFE state of alpha-lactalbumin shows little chemical shift dispersion of side-chain resonances. Extensive line broadening in the nuclear magnetic resonance spectra, characteristic of slow conformational averaging in the A state, is, however, much reduced in the TFE state. The line narrowing observed in the TFE state has made it possible to obtain directly sequence-specific assignments for about 25% of the 123 residues of bovine alpha-lactalbumin in 50% (v/v) TFE. Two helices are amongst regions of structure so far identified from short-range backbone nuclear Overhauser enhancement (NOE) connectivities in two-dimensional spectra of the TFE state. One of the helices (residues 86 to 96) corresponds to the C-helix in the native structure. The other (residues 35 to 41) corresponds, however, to a region of the sequence that is not helical in the native state. The partially folded state of alpha-lactalbumin formed in TFE, therefore, supports both native and non-native secondary structure in the absence of persistent long-range tertiary structure.
Alexandrescu
A T
AT
Oxford Centre for Molecular Sciences, University of Oxford, England.
Ng
Y L
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Dobson
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CM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006168:Guinea Pigs; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry* / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects*; D017510:Protein Folding; D014270:Trifluoroethanol / Q000494:pharmacology*
D014270:Trifluoroethanol; D007768:Lactalbumin