Specific Structure Appears at the N terminus in the Sub-millisecond Folding Intermediate of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein

Wu, Ying; Vadrevu, Ramakrishna; Yang, Xiao-Yan; Matthews, C. Robert

doi:10.1016/j.jmb.2005.06.006

Cited by 15 publications

(20 citation statements)

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“…Protein expression and purification followed standard protocols described in ref. 18. The three intrinsic cysteines present in wild-type ␣TS were not mutated, leaving the variants with four cysteine residues.…”

Section: Methodsmentioning

confidence: 99%

“…These common features in the folding mechanisms are consistent with an important role for topology in dictating the essential features of the energy landscape. However, the structured regions of these intermediates for ␣TS and IGPS, as determined from HX protection experiments (16) and mutational analysis (17,18), vary significantly. Clearly, the sequences of these proteins dictate the structures of these crucial partially folded states.…”

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confidence: 99%

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Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

Kondrashkina

Kayatekin

et al. 2008

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

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The earliest kinetic folding events for (␤␣)8 barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (␤␣) 8 barrel protein.Within 150 s, the ␣-subunit of Trp synthase (␣TS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 s, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.FRET ͉ microsecond mixing ͉ misfolding ͉ small-angle x-ray scattering T he funnel shape of typical protein folding-energy landscapes suggests that sequences have evolved to minimize energetic and topological frustration (1, 2). This view is supported by the twostate folding of many small proteins (3) and the significant correlation of their folding rates with native topology (4, 5). Morecomplex mechanisms, typical of larger proteins, often place intermediates on a progressive path to the native conformation. Surprisingly, a few kinetic studies (6-9) and simulations (10) have revealed that off-pathway intermediates can be populated during the refolding of single-domain globular proteins (11). Delineating the relative contributions of sequence and topology to biases in the energy landscape that lead to these kinetic traps offers potential insights into factors responsible for protein misfolding and, potentially, for numerous devastating pathologies (12).(␤/␣) 8 TIM barrels (named after triosephosphate isomerase) are one of the most common motifs in biology (13) and are of particular interest in examining the factors leading to off-pathway folding. They constitute the most common structural class of substrates of the chaperonin GroEL, which captures and sequesters partially folded proteins to facilitate their folding to the native state (14). Although the specific partially folded forms of the Escherichia coli TIM barrels responsible for binding to GroEL are not known, in vitro studies suggest two potential candidates. The folding reactions of three TIM barrels of very low sequence identity, IOLI (a protein of unknown function corresponding to the ioll gene in Bacillus subtilis), IGPS (indole-3-glycerol phosphate synthase), a...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

Kondrashkina

Kayatekin

et al. 2008

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

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“…An extensive mutational analysis of the equilibrium intermediate for αTS 54,55 demonstrated that a tightly-packed Nterminal region, (βα) [1][2][3][4] , is not tightly coupled to a molten globule-like C-terminal region containing (βα) [5][6][7][8] . This differential behavior was attributed to misfolding at the boundaries of the (βα) [1][2][3][4] region that precluded propagation to the C-terminal region in the I1 intermediate.…”

Section: Why Are the On-and Off-pathway Intermediates For (βα) 8 Barrmentioning

confidence: 99%

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

Rao

Forsyth

et al. 2007

Journal of Molecular Biology

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The structures of partially-folded states appearing during the folding of a (βα) 8 TIM barrel protein, the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō-model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα) 4 region, modest protection in the neighboring (βα) 1-3 and (βα) 5 β 6 segments and no significant protection in the remaining N-and Cterminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα) 2-5 β 6 region after 5 s of folding demonstrates that these species represent kinetically-distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C α Gō-model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of structure offering protection against exchange in the on-pathway intermediate (s). Because the native-centric Gō-model simulations do not explicitly include sequencespecific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō-model simulation.

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“…The average value of the half-life is 11 h. The intermediate was found to be unexpectedly stable when compared with typical folding intermediates with much shorter half-lives on the microsecond-to-millisecond timescale. 3,[5][6][7][8][9][10][11][12][13] These results indicate that the transition from the intermediate to the native state is highly cooperative and requires gross structural rearrangement. (Table 1), compared to typical folding intermediates.…”

Section: Noesy Spectra Of the Kinetically Trapped Intermediatementioning

confidence: 99%

“…However, it is difficult to experimentally determine the structural details of these intermediates because of their instability, small population size, and transient nature. 3,[5][6][7][8][9][10][11][12][13] In some cases, it has been indicated that appropriate packing in the hydrophobic core of proteins is critical for efficient folding; insufficient burial and packing of core residues lead to the accumulation of folding intermediates. [10][11][12][13][14][15][16][17] In other cases, it has been shown that interactions formed on protein surfaces are important for efficient folding.…”

Section: Introductionmentioning

confidence: 99%

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

Sugimoto

Noda

Segawa

2011

Journal of Molecular Biology

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Specific Structure Appears at the N terminus in the Sub-millisecond Folding Intermediate of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein

Cited by 15 publications

References 60 publications

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

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