Structure
file:1tim.png, Triose-phosphate isomerase (TIM) isolated from chicken muscles (), the archetypal TIM barrel enzyme. (A) Cartoon representation of the TIM barrel structure. α-helices are colored teal, β-strands are colored orange, and loops are colored green. Note that the C-terminal ends of β-strands are depicted with arrowheads. (B) Core and pore regions are highlighted. Amino acid residues belonging to the pore are colored blue. Amino acid residues belonging to the core are colored orange. Note that the TIM barrel is depicted in a ''top-down'' view, where the C-terminal ends of the β-barrel are pointed towards the reader., 300x300px, alt= file:TIM topology.png, TIM barrel topology. α-helices are colored teal, loops are colored green, and β-strands are colored in two shades of orange. Lighter shades indicate residues pointing ''inward'', towards the barrel pore. Darker shades indicate residues pointing ''outward'', towards the barrel core. Cyan lines depict an example backbone β-barrel hydrogen bonding network. Note that side-chain hydrogen bonding networks are not depicted here. Interior β-barrel residues (pore residues) display a 4-fold geometric symmetry, despite emerging from an 8-strand β-barrel. This symmetry is illustrated as two example "layers" in red and blue. Each layer contains 4 residues that point towards the pore, and lie on the same plane perpendicular to the barrel axis. The shear number for TIM barrels is always 8, and is illustrated in magenta. Some TIM barrels naturally adopt, or are designed to adopt, two or four-fold symmetry. Example asymmetric units are also highlighted. This figure has been adapted with permission from previously published work., 300x300px, alt=, leftTopology
The TIM barrel gets its name from the enzyme triose-phosphate isomerase (TIM), which was the first protein possessing the fold to be Protein crystallization, crystallized. TIM barrels contain 200-250 amino acid residues, folded into 8 alpha helices (α-helices) and 8Core 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 areTIM barrel stabilizing elements
file:1p1x_saltbridges.png, Example salt bridge network in 2-deoxyribose-5-phosphate aldolase (). Interactions are shown as cyan dashed lines. Polar residues are colored green. Polar amino acids aspartate (D), glutamate (E), lysine (K), and arginine (R), are shown here., 300x300px Salt bridge (protein and supramolecular), 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
file:independent_domains.png, Examples of structural inserts at TIM barrel loop and N/C-terminal regions. (A) The ''Bacillus subtilis'' Orotidine 5′-monophosphate decarboxylase (). Orotidine 5'-monophosphate is colored green. α-helical inserts are colored teal. The catalytic arginine residue (R215) is displayed as sticks. (B) ''Mycobacterium tuberculosis'' bifunctional histidine/tryptophan biosynthesis isomerase (PriA) (). CdRP, the product of the TrpF reaction, is colored green. β-strand/loop interchangeable structures are colored orange. (C) ''Lactococcus lactis'' dihydroorotate dehydrogenase A (DHODA) (). β-strands forming a sheet are colored orange. Extended loops are colored green. The cavity formed by these structures is displayed as a blue mesh. The product orotate is colored magenta. the cofactor FMN is colored pink. (D) ''Methylophilus methylotrophus'' trimethylamine dehydrogenase (). The Rossmann fold domain is colored according to secondary structural elements. Cofactor FMN is colored magenta. The Fe-4Ssup>+ is colored red. Note that substrate/product were not crystallized., 400x400px The N/C-terminal and loop regions on TIM barrel proteins are capable of hosting structural inserts ranging from simple secondary structural motifs to complete domains. These domains aid in substrate recognition and catalytic activity. Four diverse examples of TIM barrels containing additional motifs and domains are discussed below. ''Bacillus subtilis''Folding mechanisms
The conservation of the TIM barrel fold is mirrored by the conservation of its equilibrium and kinetic folding mechanisms in bacterial paralogs with phylogenetically distinct lineages. Chemical denaturation of several natural and 2 designed TIM barrel variants invariably involves a highly populated equilibrium intermediate. The kinetic intermediates that appear after dilution from highly denaturing solutions involve an early misfolded species that must at least partially unfold to access the productive folding pathway. The rate-limiting step in folding is the closure of the 8-stranded β-barrel, with the preceding, open barrel form corresponding to the equilibrium intermediate. Native-centric molecular dynamics simulations recapitulate the experimental results and point the way to testable computational models for complex folding mechanisms.Conserved fitness landscapes
TIM barrel proteins possess an unusually high sequence plasticity, forming large families of orthologous and paralogous enzymes in widely divergent organisms. This plasticity suggests a sequence landscape that allows for protein adaptation to a variety of environmental conditions, largely independent of phylogenetic history, while maintaining function. A deep mutational scanning approach and a competition assay was used to determine the fitness of all possible amino acid mutants across positions in 3 hyperthermophilic indole-3-glycerolphosphate synthase (IGPS) TIM barrel enzymes in supporting the growth of a yeast host lacking IGPS. Although the 2 bacterial and 1 archaeal IGPS enzymes were only 30-40% identical in sequence, their fitness landscapes were strongly correlated: the same amino acids at the same positions in the three different proteins had very similar fitness. The correlation can be thought of as the conservation of the fitness landscape for a TIM barrel enzyme across evolutionary time.Loop regions
Of the approximately 200 residues required to fully form a TIM barrel, about 160 are considered structurally equivalent between different proteins sharing this fold. The remaining residues are located on the loop regions that link the helices and strands; the loops at the C-terminal end of the strands tend to contain theEvolution and origins
file:SsIGPS reaction coordinates.png, 300px, left, The reaction coordinate diagram for SsIGPS at pH 7.8 and 25°C. The refolding reaction begins in the unfolded, ''U'' state, initially misfolds to the ''IBP'' intermediate state, partially unfolds to reach the ''IA'' intermediate state whose conversion to the subsequent ''IB'' intermediate state is rate-limiting. The final step is the conversion of ''IB'' to the native state, N. The ''IA'' and ''IB'' kinetic intermediates correspond to the intermediate observed in equilibrium unfolding studies. The ordinate represents the free energy of each state in the folding reaction mechanism in kcal mol −1. The abscissa represents the dependence of the difference in free energy between 2 states on the denaturant concentration and is proportional to the change in buried surface, referenced to the ''U'' state. The kinetic folding mechanism, illustrating the flow of the unfolded protein to the native conformation is shown beneath the reaction coordinate diagram. file:IGPS fitness landscapes.png, 300px, Experimentally derived fitness landscapes mapped from point mutations represent single steps from WT sequence. Despite significant divergence of WT in sequence space, the fitness landscapes of IGPS orthologues remain correlated (dashed lines). Rather than traditional two-dimensional heatmaps, fitness values are displayed on a three-dimensional pinwheel, highlighting the wide range of possible fitness effects of a single sequence step. The profiles of the pinwheels are similar, indicating the correlation of fitness landscapes, even if WT sequences (centers of the wheels) are only 40% identical and widely separated. Principal component analysis demonstrates a correlation between experimental fitness landscapes and amino-acid preferences in evolved sequences. The predominant theory for TIM barrel evolution involves gene duplication and fusion, starting with a half- barrel that eventually formed a full TIM barrel. Multiple studies support the theory of divergent evolution from a single ancestor, and are discussed below.Evolution from a common ancestor
In the early 1990s, it was noted that all TIM barrel structures solved at the time were enzymes, indicating divergence from a common ancestor. Further, all TIM barrels possessed active sites at the C-terminal end of β-barrels. suggested that A common phosphate binding site, formed by a small α-helix and TIM barrel loops-7/8, strongly indicated divergent evolution. Further studies of these phosphate groups, concluding that 12 of 23 SCOP TIM barrel families diverged from a common ancestor. Similarly there were hints for common ancestry for 17 of the 21Origin through gene duplication and domain fusion
file:TIM_duplication.png, 300px, Model for the evolution of TIM barrels through gene duplication and domain fusion, as proposed by Lang ''et al''. This model described the evolution of enzymes HisA and HisF of the histidine biosynthesis pathway. Two gene duplication steps are thought to have occurred. The first gene duplication resulted in two half-barrels that later fused and evolved into an ancestral TIM barrel. The second gene duplication event lead to diversification, and the evolution of different TIM barrel enzymes catalyzing different reactions. Many TIM barrel proteins possess 2-fold, 4-fold or 8-fold internal symmetry, suggesting that TIM barrels evolved from ancestral (βα)4, (βα)2, or βα motifs through gene duplication and domain fusion. A good example of 2-fold internal symmetry is observed in the enzymes ProFAR isomerase (HisA) and imidazole glycerol phosphate synthase (HisF) of the ''Thermotoga maritima'' histidine biosynthesis pathway. They catalyze 2 successive reactions in the pathway, possess 25% sequence homology, and possess root-mean-square deviations (RMSDs) between 1.5-2 Å, suggesting divergence from a common ancestor. More interestingly, the loops on the C terminal ends of both HisA and HisF showed a twofold repeated pattern, suggesting that their common ancestor also possessed 2-fold internal symmetry. Using these observations, a model was constructed for the evolution of the TIM barrels. An ancestral half-barrel would have undergone a gene duplication and fusion event, resulting in a single protein containing two half-barrel domains. Structural adaptations would have occurred, resulting in the merging of these domains to form a closed β-barrel, and forming an ancestral TIM barrel. Functional adaptations would have also occurred, resulting in the evolution of new catalytic activity at the C terminal end of the β-barrel. At this point, the common ancestor of HisA and HisF would have undergone a second gene duplication event. Divergent evolution of the duplicated genes of the ancestral TIM barrel would have resulted in the formation of HisA and HisF. Interestingly, this evolutionary model has been experimentally validated using rational protein design and''De novo'' TIM barrel design
300px, sTIM-11, the first successful ''de novo'' TIM barrel design. The asymmetric (αβ)2 units are colored distinctly, highlighting the internal 4-fold symmetry. The TIM barrel fold has been a long-standing target for ''de novo'' protein designers. As previously described, numerous TIM barrels have been successfully designed based on preexisting natural half-barrels. In contrast, the ''de novo'' design of TIM barrels occurred in incremental steps over a period of 28 years. The Octarellin series of proteins (Octarellin I→VI) were the first attempts to create a ''de novo'' TIM barrel. As the field of protein design was still in its infancy, these design attempts were only met with limited success. Although they displayed circular dichroism spectra consistent with αβ proteins and some cooperative folding characteristics, all Octarellin series peptides were insoluble, and had to be resolubilized from inclusion bodies for further characterization. Interestingly, Octarellin V.1 displayed a Rossmann-like fold under co-crystal conditions. The Symmetrin series of proteins (Symmetrin-1→4) displayed more favorable biophysical characteristics. Symmetrin-1 was readily soluble, displayed circular dichroism spectra consistent with αβ proteins, and displayed excellent cooperative unfolding and refolding characteristics. Despite these advances, all proteins in this family displayed molten characteristics when analyzed using NMR ( nuclear magnetic resonance), and further work to solve their structures could not be pursued. Proteins of the sTIM series represented the first successful ''de novo'' TIM barrel design. sTIM-11 () was designed with an internal 4-fold symmetry, to reduce the complexity of computational design using the Rosetta software suite. Previously-derived first principles were used to delineate secondary structure topologies and lengths. sTIM-11 proved to be a highly thermostable, cooperatively folding design that adopted its intended structure.See also
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