Secondary Structure and Oligomerization Behavior of Equilibrium Unfolding Intermediates of the λ Cro Repressor

Abstract

The thermal unfolding of the wild-type Cro repressor, its disulfide-bridged mutant Cro-V55C (with the Val-55 --> Cys single amino acid substitution), and a CNBr-fragment (13-66)2 of Cro-V55C was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The combined approach reveals that thermal denaturation of Cro-WT and Cro-V55C proceeds in two steps through equilibrium unfolding intermediates. The first thermal transition of the Cro-V55C dimer involves the melting of the alpha-helices and the short beta-strand localized in the N-terminal part of the molecule. This event is accompanied by the formation of tetramers, and also impacts on the hydrogen-bonding interactions of the C-terminal beta-strands. The beta-sheet formed by the C-terminal parts of each polypeptide chain is the major structural feature of the intermediate state of Cro-V55C and unfolds during a second thermal transition, which is accompanied by the dissociation of the tetramers. Cutting of 12 amino acids in the N-terminal region is sufficient to prevent the formation of alpha-helical structure in the CNBr-fragment of Cro-V55C, and to induce tetramerization already at room temperature. The tetramers may persist over a broad temperature range, and start to dissociate only upon thermal unfolding of the beta-sheet structure formed by the C-terminal regions. The wild-type protein is a dimer at room temperature and at protein concentrations of 1.8-5.8 mg/mL. At lower concentrations, the dimers are stable until the onset of thermal unfolding, which is accompanied by the dissociation of the dimers into monomers. At higher protein concentrations, the unfolding is more complex and involves the formation of tetramers at intermediate temperatures. At these intermediate temperatures, the Cro-WT has lost all of its alpha-helical structure and also most of its native beta-sheet structure. Upon further temperature increase, a tendency for an intermolecular association of the beta-strands is observed, which may result in irreversible beta-aggregation at high protein concentrations.

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

“…Based on scanning calorimetry and circular dichroism experiments (7), the heat-induced denaturation of Cro-V55C was approximated initially by a sequential three-state model: N 2 º I 2 º U 2 . Later, it was shown (8) that the intermediate state of Cro-V55C can be approximated more accurately by assuming the formation of a tetramer (2 N 2 º I 4 º 2 U 2 ), a conclusion recently corroborated by dynamic light-scattering experiments (9).…”

“…Details regarding the sample preparation and the recording of IR spectra have been described (9). IR experiments were carried out with a 16-mg͞ml Cro-V55C solution (containing 10 mM sodium cacodylate in 2 H 2 O, pH 4.0) after hydrogen deuterium exchange was complete.…”

“…Concentrationdependent scanning calorimetry profiles show that the first thermal transition of Cro-V55C is accompanied by an oligomerization of the covalently cross-linked dimers. Conventional Fourier transform IR and dynamic light-scattering experiments showed that the first thermal transition of the Cro-V55C dimer involves the unfolding of the three ␣-helices and the short ␤-strand in the N-terminal part of the protein (9). The intermediate has a well structured intermolecular ␤-sheet domain still formed by the C-terminal parts of each polypeptide chain and associates in a tetrameric structure.…”

Binding of Pediocin PA-1 with Anionic Lipid Induces Model Membrane Destabilization

“…The major component at 1,645 cm Ϫ1 may be assigned to undefined structures. However, a contribution from another structure such as 3 10 helices (type III ␤-turn) cannot be ruled out, since it is known to absorb approximately 1,639 to 1,642 cm Ϫ1 in D 2 O (54, 65) and is commonly found in peptides and globular proteins (20,26). The shoulder located at 1,625 cm Ϫ1 is attributed to ␤-strand, the one at 1,658 cm Ϫ1 to ␣-helix, whereas the small components at 1,686 and 1,675 cm Ϫ1 can be assigned to turns.…”

“…The wave number is affected by the tyrosine local environment, being different according to whether it is exposed to water or to a hydrophobic environment. For example, it increases upon peptide unfolding (26,61). Therefore, this vibration mode may represent a marker providing information relative to the N-terminal region of pediocin.…”