Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9.

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.

Using chimeric immunity proteins to explore the energy landscape for alpha-helical protein folding.

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k(UN)(H(2)O)) approximately 2000 s(-1) at pH 7.0, 25 degrees C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta(TS)0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)(Im9), Im2 (1-64)(Im9 )and Im2 (25-44)(Im9)) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)(Im9) and Im2 (56-64)(Im9) fold in two-state transitions via transition states that are significantly less compact (beta(TS) approximately 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (beta(TS) approximately 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta(I)=0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.

Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.

Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.

Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9.

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.

The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate.

Folding of the 123 amino acid residue Greek key protein apo-pseudo azurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15 degrees C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U <==> I <==> N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I <==> U <==> N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 microseconds). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.

Kinetic studies of beta-sheet protein folding.

New studies have shown that folding of beta-sheet proteins can occur with and without intermediates, with fast to slow refolding rates and late to very late transition states. These experiments demonstrate that, despite early speculation to the contrary, beta-sheet protein folding does not appear to be fundamentally different from that of helical and mixed alpha, beta proteins.