Association-induced folding of globular proteins

Abstract

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.Protein association (aggregation) is a major biomedical and biotechnological problem. Diseases such as the amyloidoses, prion diseases, and cataracts are caused by protein association, as are several diseases involving inclusion bodies or other amorphous deposits (e. g. inclusion body myositis, light chain deposition disease, Huntington's disease) (1, 2). Formation of inclusion bodies is a common problem in the production of recombinant proteins (3-5). The storage and delivery of protein drugs are also often complicated by the association process. Protein refolding is often also accompanied by aggregation, especially at higher protein concentrations, which has been attributed to the association of partially folded intermediates (6-11).Partially folded intermediates have been detected under both transient and equilibrium protein folding conditions (12-15). The structural properties of such intermediates are diverse, ranging from substantially unfolded to almost as structured as the native state (15). We have previously established that acid-unfolded staphylococcal nuclease (pH 2.5) can be transformed into one of three partially folded intermediates, depending on the nature and concentration of anions added to neutralize the repulsive effect from the net positive charges in the acid-unfolded polypeptide chain (16-17). Such partially folded species have a strong propensity to aggregate: for both apomyoglobin and staphylococcal nuclease (SNase) we have observed that the propensity to associate decreased with increasing structural content, and the least structured intermediates were monomeric only at rather dilute concentrations.We present here results of the effect of association on the structural properties of a partially folded intermediate of SNase. MATERIALS AND METHODSSNase. SNase was grown and purified from a cloned gene kindly supplied by D. Shortle (Johns Hopkins Univ. School of Medicine). The homogeneity of the protein samples was checked electrophoretically by using the PhastSystem (Pharmacia). Protein concentrations were determined from the published molar extinction coefficient. Salt titrations at low pH were carried out by making a series of solutions of desired salt concentration and adjusting the pH value with HCl or NaOH. Samples were incubated overnight before measurements. Sodium sulfate was from Sig...

Tetramer Dissociation and Monomer Partial Unfolding Precedes Protofibril Formation in Amyloidogenic Transthyretin Variants

“…Upon aggregation the TTR partially unfolded monomeric species may reacquire structure stabilized by intermolecular interactions. Observations agreeing with this possibility were reported in a study with staphylococcal nuclease, which showed that the amount of secondary structure in a monomeric partially folded intermediate doubled upon formation of soluble aggregates and the propensity to aggregate decreased with increasing structural content of the partially folded intermediate (27).…”

Metal-triggered Structural Transformations, Aggregation, and Fibrillation of Human α-Synuclein

“…Aggregation or self-association is a characteristic property of partially folded (denatured) proteins (68 -72). It has been shown that in some cases the self-association induces additional structure and stability in the partially folded intermediates (73)(74)(75). Table I shows the far-UV CD parameters of ␣-synuclein determined at different protein concentrations and indicates that the spectrum is not affected by an ϳ170-fold increase in protein concentration, consistent with the lack of self-association up to at least 600 M. Fig.…”

Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase

“…Rigorous analysis of such a system would presumably require fitting data to a polynomial whose order would depend on the number of monomers in the aggregate; an additional vexing problem is the existence of multiple oligomeric states. The unfolding process usually exhibits dependence on the concentration of the monomer as observed with staphylococcal nuclease (61,62). Although analysis of the effect of oligomerization is a tractable problem when describing the unfolding of a dimer or a monomer that goes through a dimeric intermediate, analysis of a higher order polynomial where n is indeterminate poses a significant obstacle.…”

“…Guanidine-HCl and urea can promote aggregation in unfolding studies due to the association of an intermediate (59) or the denatured state (60). Partially folded intermediates of staphylococcal nuclease aggregate to form more structured species (61,62); partially folded conformations appear to be intermediates in the formation of most aggregates (59) and can be mistaken for kinetic folding intermediates when they form transiently (63). Rigorous analysis of such a system would presumably require fitting data to a polynomial whose order would depend on the number of monomers in the aggregate; an additional vexing problem is the existence of multiple oligomeric states.…”