Consequences of localized frustration for the folding mechanism of the IM7 protein.
ABSTRACT: In the laboratory, IM7 has been found to have an unusual folding mechanism in which an "on-pathway" intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble.
Project description:The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate.
Project description:Im7 folds via an on-pathway intermediate that contains three of the four native ?-helices. The missing helix, helix III, is the shortest and its failure to be formed until late in the pathway is related to frustration in the structure. Im7H3M3, a 94-residue variant of the 87-residue Im7 in which helix III is the longest of the four native helices, also folds via an intermediate. To investigate the structural basis for this we calculated the frustration in the structure of Im7H3M3 and used NMR to investigate its dynamics. We found that the native state of Im7H3M3 is highly frustrated and in equilibrium with an intermediate state that lacks helix III, similar to Im7. Model-free analysis identified residues with chemical exchange contributions to their relaxation that aligned with the residues predicted to have highly frustrated interactions, also like Im7. Finally, we determined properties of urea-denatured Im7H3M3 and identified four clusters of interacting residues that corresponded to the ?-helices of the native protein. In Im7 the cluster sizes were related to the lengths of the ?-helices with cluster III being the smallest but in Im7H3M3 cluster III was also the smallest, despite this region forming the longest helix in the native state. These results suggest that the conformational properties of the urea-denatured states promote formation of a three-helix intermediate in which the residues that form helix III remain non-helical. Thus it appears that features of the native structure are formed early in folding linked to collapse of the unfolded state.
Project description:The small (87-residue) alpha-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an alpha-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and varphi-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.
Project description:Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val-->Thr or Phe-->Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Phi-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (>2.5 kJ mol(-1)) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.
Project description:The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A-L19A-L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, (15)N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A-L19A-L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R(h)=25 Å) relative to the urea-denatured state (R(h)?30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.
Project description:Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.
Project description:We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Phi-values and compare the results with the experimentally determined Phi-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.
Project description:Biomolecular recognition entails attractive forces for the functional native states and discrimination against potential nonnative interactions that favor alternate stable configurations. The challenge posed by the competition of nonnative stabilization against native-centric forces is conceptualized as frustration. Experiment indicates that frustration is often minimal in evolved biological systems although nonnative possibilities are intuitively abundant. Much of the physical basis of minimal frustration in protein folding thus remains to be elucidated. Here we make progress by studying the colicin immunity protein Im9. To assess the energetic favorability of nonnative versus native interactions, we compute free energies of association of various combinations of the four helices in Im9 (referred to as H1, H2, H3, and H4) by extensive explicit-water molecular dynamics simulations (total simulated time > 300 ?s), focusing primarily on the pairs with the largest native contact surfaces, H1-H2 and H1-H4. Frustration is detected in H1-H2 packing in that a nonnative packing orientation is significantly stabilized relative to native, whereas such a prominent nonnative effect is not observed for H1-H4 packing. However, in contrast to the favored nonnative H1-H2 packing in isolation, the native H1-H2 packing orientation is stabilized by H3 and loop residues surrounding H4. Taken together, these results showcase the contextual nature of molecular recognition, and suggest further that nonnative effects in H1-H2 packing may be largely avoided by the experimentally inferred Im9 folding transition state with native packing most developed at the H1-H4 rather than the H1-H2 interface.
Project description:Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.
Project description:We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.