An Obligatory Intermediate Controls the Folding of the α-Subunit of Tryptophan Synthase, a TIM Barrel Protein

Wintrode, Patrick L.; Rojsajjakul, Teerapat; Vadrevu, Ramakrishna; Matthews, C. Robert; Smith, David L.

doi:10.1016/j.jmb.2005.01.064

Cited by 26 publications

(26 citation statements)

References 49 publications

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“…Three-state folding occurs when a native-like intermediate additionally contains some slowly repaired misfolding error. In agreement, folding intermediates for many proteins are seen to be partial replicas of the native protein, whether they are kinetically populated or not, and the kinetically blocked forms are also seen to contain some significant misfolding (Kiefhaber et al 1992;Dobson et al 1994;Elöve et al 1994;Muñoz et al 1994;Sosnick et al 1994Sosnick et al , 1996Weissman and Kim 1995;Silow and Oliveberg 1997;Bai 1999;Bilsel et al 1999;Bhuyan and Udgaonkar 2001;Capaldi et al 2002;Wallace and Matthews 2002;Krishna et al 2003aKrishna et al , 2004Bollen et al 2004;Rojsajjakul et al 2004;Religa et al 2005;Wintrode et al 2005;Nishimura et al 2006).…”

Section: Three-state Foldingmentioning

confidence: 71%

A unified mechanism for protein folding: Predetermined pathways with optional errors

Mallela

Englander

2007

Protein Science

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There is a fundamental conflict between two different views of how proteins fold. Kinetic experiments and theoretical calculations are often interpreted in terms of different population fractions folding through different intermediates in independent unrelated pathways (IUP model). However, detailed structural information indicates that all of the protein population folds through a sequence of intermediates predetermined by the foldon substructure of the target protein and a sequential stabilization principle. These contrary views can be resolved by a predetermined pathway-optional error (PPOE) hypothesis. The hypothesis is that any pathway intermediate can incorporate a chance misfolding error that blocks folding and must be reversed for productive folding to continue. Different fractions of the protein population will then block at different steps, populate different intermediates, and fold at different rates, giving the appearance of multiple unrelated pathways. A test of the hypothesis matches the two models against extensive kinetic folding results for hen lysozyme which have been widely cited in support of independent parallel pathways. The PPOE model succeeds with fewer fitting constants. The fitted PPOE reaction scheme leads to known folding behavior, whereas the IUP properties are contradicted by experiment. The appearance of a conflict with multipath theoretical models seems to be due to their different focus, namely on multitrack microscopic behavior versus cooperative macroscopic behavior. The integration of three well-documented principles in the PPOE model (cooperative foldons, sequential stabilization, optional errors) provides a unifying explanation for how proteins fold and why they fold in that way.Keywords: protein folding; misfolding; foldons; sequential stabilization; optional error; predetermined pathway; lysozyme Detailed structural information obtained from hydrogen exchange (HX) studies of cytochrome c (Cyt c) and related studies with other proteins support a classical view of protein folding in which all of the molecules in a refolding population fold through essentially the same intermediate structures (Chamberlain and Marqusee 2000;Englander 2000;Maity et al. 2005). The pathway constructs the native protein by the stepwise addition of the cooperative folding units of the native structure, called foldons. The order of pathway steps is guided by a sequential stabilization process in which prior native-like structure templates the formation of subsequent complementary structure. Thus the folding pathway is determined by the same cooperative interactions that determine the target native structure. The pathway may be linear or may branch at any step as directed by the logic of the sequential stabilization process (Krishna et al. 2006 449quite differently, in terms of independent unrelated pathways (IUP model). Proteins can fold in a simple two-state way with only the unfolded (U) and native (N) states significantly populated, or in a multistate way with one or more intermediates (...

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Section: Three-state Foldingmentioning

confidence: 71%

A unified mechanism for protein folding: Predetermined pathways with optional errors

Mallela

Englander

2007

Protein Science

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“…It was also shown that the C-terminus, residues 128-268, was either unfolded or weakly folded in this intermediate. A refolding study of the urea denatured a-subunit of tryptophan synthase using pulse-quench HX MS has shown that there is an on pathway kinetic folding intermediate that shares a similar folded protein core with the I1 equilibrium intermediate (Wintrode et al, 2005). Together the data obtained for the equilibrium and kinetic intermediates show that the latter stages of the folding reaction for the a-subunit of tryptophan synthase are under thermodynamic control (Wintrode et al, 2005).…”

Section: Protein Unfolding/refoldingmentioning

confidence: 99%

Hydrogen exchange mass spectrometry for the analysis of protein dynamics

Wales

Engen

2005

Mass Spectrometry Reviews

784

908

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Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.

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“…The analysis of different ion charge state distribution (CSD) envelopes derived from different protein conformations is one of the methods for monitoring protein structural transition by ESI-MS (Chowdhury et al 1990;Loo et al 1991). ESI-MS has proven particularly useful for detection and characterization of folding/unfolding intermediates (Grandori 2002;Haq et al 2005;Wintrode et al 2005) and for kinetic studies of protein structural transitions (Konermann 2004;Furdui et al 2006;Pan et al 2006). It has been well documented that an unfolded protein molecule can accommodate more protons upon desolvation than can a compact native protein molecule because of the increased solvent-exposed surface area (Mohimen et al 2003).…”

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confidence: 99%

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

Shashilov

Ermolenkov

et al. 2007

Protein Science

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Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolutionalternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.

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An Obligatory Intermediate Controls the Folding of the α-Subunit of Tryptophan Synthase, a TIM Barrel Protein

Cited by 26 publications

References 49 publications

A unified mechanism for protein folding: Predetermined pathways with optional errors

A unified mechanism for protein folding: Predetermined pathways with optional errors

Hydrogen exchange mass spectrometry for the analysis of protein dynamics

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

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