Mapping the Structure of Folding Cores in TIM Barrel Proteins by Hydrogen Exchange Mass Spectrometry: The Roles of Motif and Sequence for the Indole-3-glycerol Phosphate Synthase from Sulfolobus solfataricus

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 05/2007; 368(2):582-94. DOI: 10.1016/j.jmb.2007.02.027
Source: PubMed


To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.

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Available from: Jill A Zitzewitz, Nov 25, 2014
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    • "At first glance the TIM-barrel topology appears as a monodomain structure, but many biophysical measurements have highlighted discrepancies between the very complex folding pathways observed and this simple picture. Actually, (β/α)8 barrels tend to behave more like multidomain proteins, with sequential folding and unfolding of subdomain folding units [67], [76]. Explaining these hierarchical folding patterns [74], [77] requires partitioning the unfolded state between off-pathway transient intermediate species with substantial secondary structure and stability [78] and on-pathway equilibrium intermediate species [79]. "
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    ABSTRACT: The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.
    PLoS ONE 08/2013; 8(8):e71858. DOI:10.1371/journal.pone.0071858 · 3.23 Impact Factor
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    • "The equilibrium intermediate accumulates at 5 M urea and consists of two conformations termed Ia and Ib. The more unstable species (Ia) is folded in the central segment (residues 48–161) and shows little or no protection in the 1–47 and 162–220 segments [44], [45]. In addition, the 59–68 loop appears disordered both in the native state and in the intermediate. "
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    ABSTRACT: The folding pathway, three-dimensional structure and intrinsic dynamics of proteins are governed by their amino acid sequences. Internal protein surfaces with physicochemical properties appropriate to modulate conformational fluctuations could play important roles in folding and dynamics. We show here that proteins contain buried interfaces of high polarity and low packing density, coined as LIPs: Light Interfaces of high Polarity, whose physicochemical properties make them unstable. The structures of well-characterized equilibrium and kinetic folding intermediates indicate that the LIPs of the corresponding native proteins fold late and are involved in local unfolding events. Importantly, LIPs can be identified using very fast and uncomplicated computational analysis of protein three-dimensional structures, which provides an easy way to delineate the protein segments involved in dynamics. Since LIPs can be retained while the sequences of the interacting segments diverge significantly, proteins could in principle evolve new functional features reusing pre-existing encoded dynamics. Large-scale identification of LIPS may contribute to understanding evolutionary constraints of proteins and the way protein intrinsic dynamics are encoded.
    PLoS ONE 10/2012; 7(10):e48212. DOI:10.1371/journal.pone.0048212 · 3.23 Impact Factor
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    • ""SC" element of a stabilization center pair in sIGPS, "SCE" ditto in eIGPS, "SR" stabilization residue in sIGPS; see [36]. "FC" element of the folding core; see [37]. "IA" interaction with substrate; see [38]. "
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    ABSTRACT: One aim of the in silico characterization of proteins is to identify all residue-positions, which are crucial for function or structure. Several sequence-based algorithms exist, which predict functionally important sites. However, with respect to sequence information, many functionally and structurally important sites are hard to distinguish and consequently a large number of incorrectly predicted functional sites have to be expected. This is why we were interested to design a new classifier that differentiates between functionally and structurally important sites and to assess its performance on representative datasets. We have implemented CLIPS-1D, which predicts a role in catalysis, ligand-binding, or protein structure for residue-positions in a mutually exclusive manner. By analyzing a multiple sequence alignment, the algorithm scores conservation as well as abundance of residues at individual sites and their local neighborhood and categorizes by means of a multiclass support vector machine. A cross-validation confirmed that residue-positions involved in catalysis were identified with state-of-the-art quality; the mean MCC-value was 0.34. For structurally important sites, prediction quality was considerably higher (mean MCC = 0.67). For ligand-binding sites, prediction quality was lower (mean MCC = 0.12), because binding sites and structurally important residue-positions share conservation and abundance values, which makes their separation difficult. We show that classification success varies for residues in a class-specific manner. This is why our algorithm computes residue-specific p-values, which allow for the statistical assessment of each individual prediction. CLIPS-1D is available as a Web service at CLIPS-1D is a classifier, whose prediction quality has been determined separately for catalytic sites, ligand-binding sites, and structurally important sites. It generates hypotheses about residue-positions important for a set of homologous proteins and focuses on conservation and abundance signals. Thus, the algorithm can be applied in cases where function cannot be transferred from well-characterized proteins by means of sequence comparison.
    BMC Bioinformatics 04/2012; 13(1):55. DOI:10.1186/1471-2105-13-55 · 2.58 Impact Factor
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