Identification and characterization of protein folding intermediates

Gianni, Stefano; Ivarsson, Ylva; Jemth, Per; Brunori, Maurizio; Travaglini-Allocatelli, Carlo

doi:10.1016/j.bpc.2007.04.008

Cited by 69 publications

(74 citation statements)

References 61 publications

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“…Determination of the individual rate constants requires fitting the data in Fig. 2 simultaneously to the two equations that are the solutions to a general three-state kinetic scheme (15,16):…”

Section: Resultsmentioning

confidence: 99%

Malleability of folding intermediates in the homeodomain superfamily

Banachewicz

Religa

Schaeffer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two-or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two-to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.protein folding | temperature jump | diffusion collision | transition state T he nucleation-condensation mechanism was proposed from a Φ-value analysis of the folding of the protein chymotrypsin inhibitor 2 (CI2) (1, 2). The individual elements of secondary structure in CI2 are not independently stable, leading to highly cooperative formation of structure in the transition state (TS) for folding in which the helix is the best-formed region, stabilized by long-range interactions. No element of secondary structure is fully formed in the TS and its folding is kinetically two-state (3). It was hypothesized that multistate folding requires independently stable elements of structure or an unstable element of structure being stabilized by interacting with other elements. At the other extreme, the three-helix engrailed homeodomain, EnHD, is a small protein that folds ultrafast via a stable intermediate in line with the framework mechanism, whereby secondary structure forms first followed by docking of the helices (4, 5). Its folding intermediate consists of helix1 (H1) being helical but not docked on to the helix2-turn-helix3 (HTH) DNA binding motif (5, 6). The HTH motif is independently stable in the on-pathway intermediate (7). Studies of other members of the homoedomain superfamily show a slide from this multistate framework folding mechanism to two-state folding and nucleation condensation for members with less stable HTH motifs, consistent with our earlier hypoth...

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Section: Resultsmentioning

confidence: 99%

Malleability of folding intermediates in the homeodomain superfamily

Banachewicz

Religa

Schaeffer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Such low energy intermediates might aggregate through exposure of hydrophobic patches, which would explain why these intermediates are rare [1]. Nevertheless, the protein folding energy landscape is not a smooth surface, instead it is rugged and broad [2][3][4].…”

Section: Introductionmentioning

confidence: 99%

An expanded view of the protein folding landscape of PDZ domains

Hultqvist¹,

Pedersen²,

Celestine³

et al. 2012

Biochemical and Biophysical Research Communications

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Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

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“…All of the folding limbs for the fast phase converge at ∼3 × 10 5 s −1 . The slow phases exhibited rollover, characteristic of multistate kinetics (12)(13)(14).…”

Section: Resultsmentioning

confidence: 99%

“…All of the folding limbs for the fast phase converge at ∼3 × 10 5 s −1 . The slow phases exhibited rollover, characteristic of multistate kinetics (12)(13)(14).The dependence of the logarithms of the relaxation rate constants λ on [Urea] were fitted simultaneously assuming that each phase was two-state and each individual rate constant (k) had the standard linear dependence on [Urea], k ¼ k 0 expðm½urea ∕RTÞ, (Fig. 3).…”

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confidence: 99%

Folding of the Pit1 homeodomain near the speed limit

Banachewicz

Johnson

Fersht

2010

Proc. Natl. Acad. Sci. U.S.A.

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Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 μs to form a wellcharacterized intermediate, which rearranges in 17 μs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 μs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10 5 s −1 in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10 5 s −1 at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.activation energy | barrier-limited | chevron plot | dynamics T he homeodomain superfamily is comprised of small threehelical-bundle proteins (1). Their pathways of folding are among the best characterized, with information coming from the effects of point mutations on the folding kinetics of individual members and by comparison of the different members of the family with the same fold but grossly different sequences (2). The mechanism of folding slides from a clear framework mechanism for the engrailed homoeodomain (EnHD) to nucleation-condensation for c-Myb (1, 3). EnHD is an ultrafast folding protein that forms an on-pathway intermediate in 1.7 μs, which rearranges in 17 μs to the native structure. The mechanism is unusually very well-established because the structure of the intermediate has been solved by NMR; it consists of helices 2 and 3 (H2, H3) in native-like conformation with H1 being helical but undocked (4). The H2-turn-H3 motif, is in fact, a stable domain of EnHD (5). Temperature-jump experiments using both IR to monitor secondary structure formation, and Trp fluorescence for tertiary interactions have been used to follow the folding of the wild-type protein, a mutant in which the H1 does not dock at physiological ionic strength, and an H2-turn-H3 construct (5). The H2-turn-H3 domain forms both secondary and tertiary structure in 1.7 μs, and the docking of H1 takes 17 μs. Direct mea...

show abstract

Identification and characterization of protein folding intermediates

Cited by 69 publications

References 61 publications

Malleability of folding intermediates in the homeodomain superfamily

Malleability of folding intermediates in the homeodomain superfamily

An expanded view of the protein folding landscape of PDZ domains

Folding of the Pit1 homeodomain near the speed limit

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