, TIM, barrel motif is one of the most common in biology, accounting for approximately 10% of the folds in all three super-kingdoms of life. Its capacity to catalyze five of six categories of biochemical reactions, including many essential metabolic reactions, implies that this motif arose early in evolution and flourished because of its catalytic adaptability.1-4
Examination of the structures accessible to these βα-repeat proteins shows that, although pseudo-barrels with as few as 6 and expanded barrels with as many as 9 βα repeat units exist, the most common barrels contain 8 repeat units.3
The repeats are sequentially-ordered into parallel-stranded arrays, with hydrogen bonds between strands 1 and 8 forming a cylindrical barrel. The anti-parallel amphipathic helices, alternating in the sequence with the strands, form a continuous shell around the largely nonpolar strands. The active sites are invariably defined by the loops at the C-termini of the strands, enabling the conservation of the structural core while offering a multitude of possibilities for substrate and cofactor recognition and for catalytic function.5
In this sense, (βα)8
barrels are analogous to antibodies, where the anti-parallel β sandwich cores provide a stable platform for the somatic mutations in the loops that enable exquisite recognition of a plethora of antigenic determinants.
Although the (βα)8
motif is a single structural domain, its folding free energy surface is more complex than that expected for a simple, highly-cooperative unfolding transition to the denatured state. Studies on several (βα)8
barrel proteins, including the alpha subunit of tryptophan synthase from E. coli
the indole-3-glycerol phosphate synthase from S. solfataricus
and from E. coli
and the N-(5′-phosphoribosyl)anthranilate isomerase from E. coli
have been shown to have stable intermediates with substantial thermodynamic stability that retain significant secondary structure (~50%) when probed by far-UV CD. Kinetic studies have shown that the folding of this intermediate to the native state can be the rate-limiting step in a complex reaction scheme.7,9,10
Given the common occurrence of the equilibrium intermediate and its crucial role in folding, this species has been the subject of several fragmentation studies of its structure.
Early fragment complementation studies on αTS and PRAI were interpreted to mean that these proteins fold via a 6+2 model, with the (βα)1-6
folding nucleus corresponding to the equilibrium intermediate for both proteins.11-13
However, a more comprehensive fragmentation analysis of αTS demonstrated that the equilibrium unfolding properties of this TIM barrel are more complex than revealed by the chemical denaturation experiment. The results led to the hypothesis that folding involves the modular assembly of βαβ supersecondary structural elements.14
N-terminal fragments containing as few as 4 βα repeats can fold cooperatively. The addition of the β5
module confers enhanced stability, consistent with a refined (4+2)+2 model. A more recent characterization of a series of fragments and circularly permuted variants of PRAI, combined with molecular dynamics simulations, led to the proposal of a 5+1+2 folding model in which the (βα)1-6
segment corresponds most closely to the equilibrium intermediate.15
By contrast, the recovery of catalytic activity upon the complementation of the N- and C-terminal halves of the HisF barrel16
and the retention of structure and stability in the C-terminal half barrel17
support a 4+4 mechanism of folding. The (βα)5-8
segment would presumably serve as the platform defining the equilibrium intermediate observed in a chemical denaturation experiment (Z. Gu and C. R. Matthews, unpublished results). These fragmentation studies have provided strong evidence for the modular assembly of (βα)8
proteins. Relevant to the present study, the different models for assembly suggest that the structures of the corresponding intermediates are strongly influenced, if not controlled, by the amino acid sequence.
Hydrogen exchange mass spectrometry (HX-MS) has recently been used to obtain structural information on folding intermediates of several TIM barrel proteins, including αTS,18
the multimeric aldolase19
and triose phosphate isomerase, TIM.20
Brief exposure of transient or stable partially-folded forms to 2
O, quenching with acid and digestion with pepsin enables the identification by MS of segments where the protection of amide hydrogens against solvent exchange implies structure. The most stable core of the equilibrium unfolding intermediate for αTS observed by HX-MS at 3 M urea is comprised of the (β/α)1-3
segment, consistent with previous fragmentation studies and a (4+2)+2 folding model.18
Although the dimeric rabbit TIM does not appear to populate a stable intermediate under equilibrium conditions, a partially-folded state involving the (βα)5-8
segment was observed in HX-MS studies of a late intermediate in the refolding reaction.20
The results were interpreted in terms of a 4+4 C-terminus driven folding mechanism, similar to HisF.16
Two rare partially-folded states in equilibrium with native dimeric yeast TIM were detected by a complementary technique, misincorporation proton-alkyl exchange (MPAX).21
By monitoring the transient exposure of cysteines to water-soluble sulfhydryl reagents, the most stable core, i.e. segments resistant to modification, was shown to involve strands β2
. The presumed folding model is (1+(3)+2)+2 and is similar to that for αTS. The various combinations of βα modules observed to define the structures of partially-folded states in TIM barrel proteins show that the conservation of the discrete thermodynamic state for the intermediate detected by chemical denaturation has not been realized in the conservation of its structure nor, possibly, in its singularity.
To further explore the relationship between the motif, the amino acid sequence and the structure of the stable intermediate in (βα)8 barrels, the secondary structures of the pair of corresponding intermediates in sIGPS have been examined by HX-MS. The locations of the elements of secondary structure in the two equilibrium intermediates for sIGPS differ from that for the single species in αTS, consistent with the hypothesis that the structures of these intermediates are less conserved than their existence. Strikingly, the protection patterns in both sIGPS and αTS appear to correlate with the presence of hydrophobic clusters comprised of branched aliphatic side chains in the native conformation.