The addition of HCI, at low ionic strength, to the native state of apomyoglobin, fi-lactamase, and cytochrome c caused these proteins to adopt an essentially fully unfolded conformation in the vicinity of pH 2. However, contrary to expectation, the addition of further acid resulted in refolding to a compact conformation with the properties of a molten globule. The major factor responsible for the refolding is believed to be the binding of the anion, which minimizes the intramolecular charge repulsion that initially brought about the unfolding.One of the oldest known methods of denaturing proteins is by the addition of acids. Reports on the conformational state of the denatured protein have varied from apparently fully unfolded to substantial remaining structure. We have recently reported that 83-lactamase I from Bacillus cereus adopts a relatively fully unfolded conformational state at pH 2 at low ionic strength but on the addition of salts converts to a compact, molten globule-like state (1). We now show for several proteins that, if one starts with a native protein at low ionic strength and lowers the pH by adding HCI, the protein will initially change to a relatively fully unfolded conformation, typically in the vicinity of pH 2, and then, as the acid concentration increases, it will change into a compact conformation containing substantial secondary structure.We use the following experimental criteria to define the protein conformational states expected in the low pH region. (i) The native state is characterized by its near-UV and far-UV circular dichroism (CD) spectra as determined at pH 7, a tryptophan fluorescence emission spectrum with Amax as determined at pH 7 (usually in the vicinity of 335 nm), and the Stokes radius as determined at pH 7 using dynamic light scattering or gel-exclusion chromatography. (ii) The acidunfolded state, UA, has a CD spectrum similar to that of the protein unfolded in 5 or 6 M guanidine hydrochloride (Gdn HCl) at neutral pH, a tryptophan fluorescence Amax around 350 nm, and a Stokes radius .2 times that of the native state (for proteins without disulfide bonds) (1). (iii) State A is a compact state (as determined from its hydrodynamic radius) with substantial secondary structure (as determined by far-UV CD) but little or no native-like tertiary structure (as determined by near-UV CD), and tryptophan fluorescence emission with a Amax similar to that of the native state. State A has the properties ascribed to a compact state that has been called a molten globule (1-3). We believe that this species, as described above, may be considered to consist of an overall fold similar to that of the native state in which structural elements, typically secondary structural units, have been pulled apart somewhat, leading to some solvent penetration and enhanced side-chain mobility. In some cases (e.g., apomyoglobin, discussed below), part of the molecule (e.g., the C-terminal region) may be fully unfolded. MATERIALS AND METHODSMaterials. /3-Lactamase I from B. cereus was prepared as ...

A systematic investigation of the effect of acid on the denaturation of some 20 monomeric proteins indicates that several different types of conformational behavior occur, depending on the protein, the acid, the presence of salts or denaturant, and the temperature. Three major types of effects were observed. Type I proteins, when titrated with HCl in the absence of salts, show two transitions, initially unfolding in the vicinity of pH 3-4 and then refolding to a molten globule-like conformation, the A state, at lower pH. Two variations in this behavior were noted: some type I proteins, when titrated with HCl in the absence of salts, show only partial unfolding at pH 2 before the transition to the molten globule state; others of this class form an A state that is a less compact from of the molten globule state. In the presence of salts, these proteins transform directly from the native state to the molten globule conformation. Type II proteins, upon acid titration, do not fully unfold but directly transform to the molten globule state, typically in the vicinity of pH 3. Type III proteins show no significant unfolding to pH as low as 1, but may be caused to behave similarly to type I in the presence of urea. Thus, the exact behavior of a given protein at low pH is a complex interplay between a variety of stabilizing and destabilizing forces, some of which are very sensitive to the environment. In particular, the protein conformation is quite sensitive to salts (anions) that affect the electrostatic interactions, denaturants, and temperature, which cause additional global destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near-and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCI results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye I-anilino-%naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states.A significant decrease in a-helix structure, with a corresponding increase in 6 or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.

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