Dataset Information

Refolding the engrailed homeodomain: structural basis for the accumulation of a folding intermediate.

ABSTRACT: The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in approximately 15 mus. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to why rearrangements of intermediates occur slowly. To address this problem, we performed 46 all-atom molecular dynamics refolding simulations in explicit water, for a total of 15 micros of simulation time. The simulations started from an intermediate state structure that was generated in an unfolding simulation at 498 K and was then quenched to folding-permissive temperatures. The protein refolded successfully in only one of the 46 simulations, and in that case the refolding pathway mirrored the unfolding pathway at high temperature. In the 45 simulations in which the protein did not fully fold, nonnative salt bridges trapped the protein, which explains why the protein folds relatively slowly from the intermediate state.

SUBMITTER: McCully ME

PROVIDER: S-EPMC2931720 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Refolding the engrailed homeodomain: structural basis for the accumulation of a folding intermediate.

McCully Michelle E ME Beck David A C DA Fersht Alan R AR Daggett Valerie V

Biophysical journal 20100901 5

The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in approximately 15 mus. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to wh ...[more]

PMID: 20816076

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Project description:The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate.

Dataset Information

Refolding the engrailed homeodomain: structural basis for the accumulation of a folding intermediate.

Publications

Refolding the engrailed homeodomain: structural basis for the accumulation of a folding intermediate.

Similar Datasets

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