TIM barrel

The TIM barrel (triose-phosphate isomerase), also known as an alpha/beta barrel, is a conserved protein fold consisting of eight alpha helices (α-helices) and eight parallel beta strands (β-strands) that alternate along the peptide backbone. The structure is named after triose-phosphate isomerase, a conserved metabolic enzyme. TIM barrels are ubiquitous, with approximately 10% of all enzymes adopting this fold. Further, five of seven enzyme commission (EC) enzyme classes include TIM barrel proteins. The TIM barrel fold is evolutionarily ancient, with many of its members possessing little similarity today, instead falling within the twilight zone of sequence similarity. The inner beta barrel (β-barrel) is in many cases stabilized by intricate salt-bridge networks. Loops at the C-terminal ends of the β-barrel are responsible for catalytic activity while N-terminal end loops are important for the stability of the TIM-barrels. Structural inserts ranging from extended loops to independent protein domains may be inserted in place of these loops or at the N-terminus/C-terminals. TIM barrels appear to have evolved through gene duplication and domain fusion events of half-barrel proteins, with a majority of TIM barrels originating from a common ancestor. This led many TIM barrels to possess internal symmetries. Further gene duplication events of this ancestral TIM barrel led to diverging enzymes possessing the functional diversity observed today. TIM barrels have also been a longstanding target for protein designers. Successful TIM barrel designs include both domain fusions of existing proteins and de novo designs. Domain fusions experiments have resulted in many successful designs, whereas de novo designs only yielded successes after 28 years of incremental development.

Structure

Topology The TIM barrel gets its name from the enzyme triose-phosphate isomerase (TIM), which was the first protein possessing the fold to be crystallized. TIM barrels contain 200-250 amino acid residues, folded into 8 alpha helices (α-helices) and 8 beta strand (β-strands). The β-strands are arranged into a parallel beta barrel (β-barrel), and are surrounded by the 8 α-helices. The defining property of TIM β-barrels is that they always possess a shear number of 8. The shear number is determined by picking a residue x on β-strand-1, and moving along the β-barrel, in a perpendicular direction to the direction of the strands, until residue y on the original β-strand-1 is reached. The number of residues between the start and end positions (|y−x|) is the shear number. Since the number of strands is equal to the shear number, side-chains point alternatively towards the pore and the core, giving a 4-fold symmetry. The α-helices surround and completely enclose the inner β-barrel. Short loops typically connect the α and β secondary structures, forming a (βα)8 repeat topology. In some cases, structures ranging from extended loops to independent domains may be inserted in place of these loops, or may be attached to the N/C-terminals. All TIM barrel enzymes possess catalytic sites at the C-terminal end of the β-barrel, and structural inserts present close to this end may aid in catalytic activity.

Core and pore regions TIM barrels contain two distinct buried regions, where amino acid residues are completely enveloped by their neighbors and lack access to solvent. The term 'pore' is a misnomer, as no solvent channels exist within this region. The core region consists of all residues constituting the α-β interface, and lies exterior to the central β-barrel. The pore region consists of all interior β-barrel residues, which are surrounded and enclosed by the β-barrel backbone. Due to the pleated nature of β-strands, alternate residues along a strand are almost evenly split between the pore (53%) and core (47%). For β-barrels, 95% of their core residues are buried. Only 11% of their core residues are polar, possessing an affinity for water, and possessing the ability to form hydrogen bonds or salt bridges. Similarly, 84% of β-strand pore residues are buried. However, 42% of their pore residues are polar. These residues form intricate salt bridge networks to compensate for their lack of solvent accessibility.

TIM barrel stabilizing elements

Salt bridges within TIM barrel pores are thought to contribute to the overall stability of the fold. An example of a large salt bridge network can be found in 2-deoxyribose-5-phosphate aldolase. This network was found to be conserved across the Class I aldolase family. The exact reason for the overrepresentation of polar residues and salt bridges within the pore remains unclear. One study proposes that they improve foldability rather than thermodynamic stability of TIM barrels. During the folding process, inner pore residues on β-strands would be exposed to water. Partially-folded βαβα modules, called foldons, would be energetically stabilized by polar pore residues during this stage of folding. In another study involving the S. solfataricus indole-3-glycerol phosphate synthase TIM barrel protein, a conserved βαβαβ module was found to be an essential folding template, which guided the folding of other secondary structures. β-barrel closure only occurred at the end of the folding process. In this case however, the authors credited branched aliphatic amino acids (valine, leucine, and isoleucine) for foldon stability. Another stabilizing element in TIM barrels is the beta hairpin clamp. Side chain H-bond donors at the N-termini of even-numbered β-strands often form H-bonds with main chain amide hydrogens in preceding odd-numbered β-strands. These clamps (or hydrophobic side chain bridge analogs) are conserved in 3 indole-3-glycerolphosphate synthase TIM barrel orthologs from the bacterial and archaeal kingdoms, implying they arose in their last common ancestor and have been preserved for over a billion years.

Structural inserts

… excerpt ends here. Continue reading the full article.