View source for Biology:TIM barrel

{{short description|Protein fold}}
{{Pfam box
|image=8tim TIM barrel topview.png
|caption=Top view of a triosephosphateisomerase (TIM) barrel ({{PDB|8TIM}}), colored from blue ([[Biology:N-terminus|N-terminus]]) to red (C-terminus).
|Name=Aldolase-type TIM barrel
|Symbol=Aldolase_TIM
|Pfam_clan=CL0036
|InterPro=IPR013785
|CATH=8tim
|SCOP=8tim
}}

The '''TIM barrel''' (triose-phosphate isomerase), also known as an '''alpha/beta barrel''',<ref name="voet">{{cite book|title=Biochemistry|edition=4th| vauthors = Voet D, Voet JG |publisher=John Wiley & Sons, Inc.|year=2011|isbn=978-0470-91745-9|chapter=Chapter 8. Three-Dimensional Structures of Proteins}}</ref>{{rp|252}} is a [[Biology:Conserved sequence|conserved]] [[Biology:Protein folding|protein fold]] consisting of eight alpha helices (α-helices) and eight parallel beta strands (β-strands) that alternate along the [[Biology:Tertiary structure|peptide backbone]].<ref name="wierenga2001tim2">{{cite journal | vauthors = Wierenga RK | title = The TIM-barrel fold: a versatile framework for efficient enzymes | journal = FEBS Letters | volume = 492 | issue = 3 | pages = 193–8 | date = March 2001 | pmid = 11257493 | doi = 10.1016/s0014-5793(01)02236-0 | s2cid = 42044123 | doi-access = free }}</ref> The structure is named after triose-phosphate isomerase, a conserved metabolic [[Biology:Enzyme|enzyme]].<ref name="banner1975structure2">{{cite journal | vauthors = Banner DW, Bloomer AC, Petsko GA, Phillips DC, Pogson CI, Wilson IA, Corran PH, Furth AJ, Milman JD, Offord RE, Priddle JD, Waley SG | display-authors = 6 | title = Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5 angstrom resolution using amino acid sequence data | journal = Nature | volume = 255 | issue = 5510 | pages = 609–14 | date = June 1975 | pmid = 1134550 | doi = 10.1038/255609a0 | s2cid = 4195346 }}</ref> TIM barrels are ubiquitous, with approximately 10% of all [[Biology:Enzyme|enzyme]]s adopting this fold.<ref name="jansen2000analysis">{{cite journal | vauthors = Jansen R, Gerstein M | title = Analysis of the yeast transcriptome with structural and functional categories: characterizing highly expressed proteins | journal = Nucleic Acids Research | volume = 28 | issue = 6 | pages = 1481–8 | date = March 2000 | pmid = 10684945 | doi = 10.1093/nar/28.6.1481 | pmc = 111042 | doi-access = free }}</ref> Further, five of seven [[Biology:Enzyme Commission number|enzyme commission]] (EC) enzyme classes include TIM barrel proteins.<ref name="nagano1999barrel">{{cite journal | vauthors = Nagano N, Hutchinson EG, Thornton JM | title = Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels | journal = Protein Science | volume = 8 | issue = 10 | pages = 2072–84 | date = October 1999 | pmid = 10548053 | doi = 10.1110/ps.8.10.2072 | pmc = 2144152 }}</ref><ref name="webb1992enzyme">{{cite book| vauthors = Webb EC |title=Enzyme nomenclature: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes|date=1992|publisher=Academic Press|isbn=978-0-12-227164-9|issue=Ed. 6}}</ref> The TIM barrel fold is [[Biology:Conserved sequence|evolutionarily ancient]], with many of its members possessing little [[Sequence alignment|similarity]] today,<ref name="nagano2002one">{{cite journal | vauthors = Nagano N, Orengo CA, Thornton JM | title = One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions | journal = Journal of Molecular Biology | volume = 321 | issue = 5 | pages = 741–65 | date = August 2002 | pmid = 12206759 | doi = 10.1016/s0022-2836(02)00649-6 }}</ref> instead falling within the ''twilight zone'' of sequence similarity.<ref name="livesay2005evolutionary">{{cite journal | vauthors = Livesay DR, La D | title = The evolutionary origins and catalytic importance of conserved electrostatic networks within TIM-barrel proteins | journal = Protein Science | volume = 14 | issue = 5 | pages = 1158–70 | date = May 2005 | pmid = 15840824 | doi = 10.1110/ps.041221105 | pmc = 2253277 }}</ref><ref name="chung1996structural">{{cite journal | vauthors = Chung SY, Subbiah S | title = A structural explanation for the twilight zone of protein sequence homology | journal = Structure | volume = 4 | issue = 10 | pages = 1123–7 | date = October 1996 | pmid = 8939745 | doi = 10.1016/s0969-2126(96)00119-0 | doi-access = free }}</ref>

The inner [[Biology:Beta barrel|beta barrel]] (β-barrel) is in many cases stabilized by intricate [[Chemistry:Salt bridge (protein and supramolecular)|salt-bridge networks]].<ref name="vijayabaskar2012insights">{{cite journal | vauthors = Vijayabaskar MS, Vishveshwara S | title = Insights into the fold organization of TIM barrel from interaction energy based structure networks | journal = PLOS Computational Biology | volume = 8 | issue = 5 | pages = e1002505 | date = 2012 | pmid = 22615547 | doi = 10.1371/journal.pcbi.1002505 | pmc = 3355060 | bibcode = 2012PLSCB...8E2505V | doi-access = free }}</ref> Loops at the C-terminal ends of the β-barrel are responsible for catalytic activity<ref name="farber1990evolution">{{cite journal | vauthors = Farber GK, Petsko GA | title = The evolution of alpha/beta barrel enzymes | journal = Trends in Biochemical Sciences | volume = 15 | issue = 6 | pages = 228–34 | date = June 1990 | pmid = 2200166 | doi = 10.1016/0968-0004(90)90035-A }}</ref><ref name="Farber1995structure2">{{cite journal | vauthors = Reardon D, Farber GK | title = The structure and evolution of alpha/beta barrel proteins | journal = FASEB Journal | volume = 9 | issue = 7 | pages = 497–503 | date = April 1995 | pmid = 7737457 | doi = 10.1096/fasebj.9.7.7737457 | s2cid = 23208817 }}</ref> while N-terminal end loops are important for the stability of the TIM-barrels. Structural inserts ranging from extended loops to independent [[Biology:Protein domain|protein domain]]s may be inserted in place of these loops or at the N-terminus/C-terminals. TIM barrels appear to have evolved through [[Biology:Gene duplication|gene duplication]] and domain fusion events of half-barrel proteins,<ref name="lang2000structural2">{{cite journal | vauthors = Lang D, Thoma R, Henn-Sax M, Sterner R, Wilmanns M | title = Structural evidence for evolution of the beta/alpha barrel scaffold by gene duplication and fusion | journal = Science | volume = 289 | issue = 5484 | pages = 1546–50 | date = September 2000 | pmid = 10968789 | doi = 10.1126/science.289.5484.1546 | bibcode = 2000Sci...289.1546L }}</ref> with a majority of TIM barrels originating from a [[Biology:Common descent|common ancestor]]. This led many TIM barrels to possess internal symmetries.<ref name="soding2006hhrep2">{{cite journal | vauthors = Söding J, Remmert M, Biegert A | title = HHrep: de novo protein repeat detection and the origin of TIM barrels | journal = Nucleic Acids Research | volume = 34 | issue = Web Server issue | pages = W137-42 | date = July 2006 | pmid = 16844977 | doi = 10.1093/nar/gkl130 | pmc = 1538828 | doi-access = free }}</ref> 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 [[Biology:Protein design|protein design]]ers. Successful TIM barrel designs include both domain fusions of existing proteins and ''de novo'' designs. Domain fusions experiments have resulted in many successful designs,<ref name="seitz2007stabilisation2">{{cite journal | vauthors = Seitz T, Bocola M, Claren J, Sterner R | title = Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels | journal = Journal of Molecular Biology | volume = 372 | issue = 1 | pages = 114–29 | date = September 2007 | pmid = 17631894 | doi = 10.1016/j.jmb.2007.06.036 }}</ref><ref name="hocker2009high2">{{cite journal | vauthors = Höcker B, Lochner A, Seitz T, Claren J, Sterner R | title = High-resolution crystal structure of an artificial (betaalpha)(8)-barrel protein designed from identical half-barrels | journal = Biochemistry | volume = 48 | issue = 6 | pages = 1145–7 | date = February 2009 | pmid = 19166324 | doi = 10.1021/bi802125b }}</ref><ref name="hocker2004mimicking">{{cite journal | vauthors = Höcker B, Claren J, Sterner R, Makar AB, McMartin KE, Palese M, Tephly TR | title = Formate assay in body fluids: application in methanol poisoning | journal = Biochemical Medicine | volume = 13 | issue = 2 | pages = 117–26 | date = June 1975 | pmid = 15539462 | doi = 10.1016/0006-2944(75)90147-7 | pmc = 534502 }}</ref><ref name="claren2009establishing2">{{cite journal | vauthors = Claren J, Malisi C, Höcker B, Sterner R | title = Establishing wild-type levels of catalytic activity on natural and artificial (beta alpha)8-barrel protein scaffolds | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 10 | pages = 3704–9 | date = March 2009 | pmid = 19237570 | doi = 10.1073/pnas.0810342106 | pmc = 2656144 | bibcode = 2009PNAS..106.3704C | doi-access = free }}</ref><ref name="bharat2008betaalpha2">{{cite journal | vauthors = Bharat TA, Eisenbeis S, Zeth K, Höcker B | title = A beta alpha-barrel built by the combination of fragments from different folds | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 29 | pages = 9942–7 | date = July 2008 | pmid = 18632584 | doi = 10.1073/pnas.0802202105 | pmc = 2481348 | bibcode = 2008PNAS..105.9942B | doi-access = free }}</ref><ref name="eisenbeis2012potential2">{{cite journal | vauthors = Eisenbeis S, Proffitt W, Coles M, Truffault V, Shanmugaratnam S, Meiler J, Höcker B | title = Potential of fragment recombination for rational design of proteins | journal = Journal of the American Chemical Society | volume = 134 | issue = 9 | pages = 4019–22 | date = March 2012 | pmid = 22329686 | doi = 10.1021/ja211657k }}</ref><ref name="fortenberry2011exploring2">{{cite journal | vauthors = Fortenberry C, Bowman EA, Proffitt W, Dorr B, Combs S, Harp J, Mizoue L, Meiler J | display-authors = 6 | title = Exploring symmetry as an avenue to the computational design of large protein domains | journal = Journal of the American Chemical Society | volume = 133 | issue = 45 | pages = 18026–9 | date = November 2011 | pmid = 21978247 | doi = 10.1021/ja210593m | pmc = 3781211 }}</ref> whereas ''de novo'' designs only yielded successes after 28 years of incremental development.<ref name="huang2016novo2">{{cite journal | vauthors = Huang PS, Feldmeier K, Parmeggiani F, Velasco DA, Höcker B, Baker D | title = De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy | journal = Nature Chemical Biology | volume = 12 | issue = 1 | pages = 29–34 | date = January 2016 | pmid = 26595462 | doi = 10.1038/nchembio.1966 | pmc = 4684731 }}</ref>

== Structure ==
thumb|'''Triose-phosphate isomerase (TIM)''' isolated from chicken muscles ({{PDB|1TIM}}), 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|thumb|'''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.<ref name=nagarajan2015design>{{cite journal | vauthors = Nagarajan D, Deka G, Rao M | title = Design of symmetric TIM barrel proteins from first principles | journal = BMC Biochemistry | volume = 16 | issue = 1 | pages = 18 | date = August 2015 | pmid = 26264284 | doi = 10.1186/s12858-015-0047-4 | pmc = 4531894 | doi-access = free }}</ref>|300x300px|alt=|left]]

=== Topology ===
The TIM barrel gets its name from the enzyme triose-phosphate isomerase (TIM), which was the first protein possessing the fold to be [[Biology:Protein crystallization|crystallized]].<ref name="banner1975structure2"/> TIM barrels contain 200-250 amino acid residues,<ref name="wierenga2001tim2"/> folded into 8 alpha helices (α-helices) and 8 beta strand (β-strands). The β-strands are arranged into a parallel [[Biology:Beta barrel|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.<ref name="wierenga2001tim2"/> 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.<ref name="murzin1994principles">{{cite journal | vauthors = Murzin AG, Lesk AM, Chothia C | title = Principles determining the structure of beta-sheet barrels in proteins. I. A theoretical analysis | journal = Journal of Molecular Biology | volume = 236 | issue = 5 | pages = 1369–81 | date = March 1994 | pmid = 8126726 | doi = 10.1016/0022-2836(94)90064-7 }}</ref> 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)<sub>8</sub> 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,<ref name="branden1991tim">{{cite journal|last1=Brändén|first1=Carl-Ivar | name-list-style = vanc |date=1991|title=The TIM barrel—the most frequently occurring folding motif in proteins|journal=Current Opinion in Structural Biology|volume=1|issue=6|pages=978–983|doi=10.1016/0959-440x(91)90094-a}}</ref> and structural inserts present close to this end may aid in catalytic activity.

=== Core and pore regions ===
TIM barrels contain two distinct [[Biology:Residue depth|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 [[Chemistry:Chemical polarity|polar]], possessing an affinity for water, and possessing the ability to form hydrogen bonds or salt bridges.<ref name="vijayabaskar2012insights" /> 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 ===
thumb|'''Example salt bridge network''' in 2-deoxyribose-5-phosphate aldolase ({{PDB|1P1X}}). 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

[[Chemistry: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 [[Biology:Deoxyribose-phosphate aldolase|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 [[Biology:Protein folding|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'' [[Biology:Indole-3-glycerol-phosphate synthase|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 ===
thumb|'''Examples of structural inserts at TIM barrel loop and N/C-terminal regions.''' '''(A)''' The ''Bacillus subtilis'' Orotidine 5′-monophosphate decarboxylase ({{PDB|1DBT}}). 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) ({{PDB|2Y85}}). 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) ({{PDB|2DOR}}). 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 ({{PDB|2TMD}}).<ref name=barber1992correlation>{{cite journal | vauthors = Barber MJ, Neame PJ, Lim LW, White S, Matthews FS | title = Correlation of x-ray deduced and experimental amino acid sequences of trimethylamine dehydrogenase | journal = The Journal of Biological Chemistry | volume = 267 | issue = 10 | pages = 6611–9 | date = April 1992 | doi = 10.1016/S0021-9258(19)50471-9 | pmid = 1551870 | doi-access = free }}</ref> The Rossmann fold domain is colored according to secondary structural elements. Cofactor FMN is colored magenta. The [4Fe-4S]<sup>+</sup> 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 [[Biology:Protein secondary structure|secondary structural motifs]] to complete [[Biology:Protein domain|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'' [[Biology:Orotidine 5'-phosphate decarboxylase|Orotidine 5'-phosphate decarboxylase]] ({{PDB|1DBT}}) is a TIM barrel protein displaying 4 α-helices in place of the βα loops typically present at the C-terminal of the β-barrel (residues 35-42, 89-91, 126-133, and 215-219). One of these helices (R215→K219) contains a conserved [[Chemistry:Arginine|arginine]]<nowiki/>residue (R215) required for interacting with a phosphate moiety on orotidine 5′-monophosphate. The other helices were not found to host residues critical for catalytic activity, and may serve in structural roles.

Mycobacterium tuberculosis bifunctional histidine/tryptophan biosynthesis isomerase (PriA) ({{PDB|2Y85}}) possesses the ability to catalyse two reactions: (i) HisA reaction: the conversion of N-[(5-phosphoribosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N-[(5-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR), and (ii) TrpF reaction: N-(5’-phosphoribosyl)-anthranilate (PRA) to 1-(O-carboxyphenylamino)- 1’-deoxyribulose-5’-phosphate (CdRP). PriA is a TIM barrel enzyme that accommodates both substrates using active site loops (loops 1, 5, and 6, extended βα loops at the C-terminal end of the β-barrel) that change conformation depending on the reactant present. Loop 1 wraps over the active site only in the presence of ProFAR. Loop5 wraps over the active site, adopting a β-sheet conformation in the presence of CdRP, or a knot-like conformation in the presence of ProFAR. Loop 6 wraps over the active site for all reactants.

''Lactococcus lactis'' [[Biology:Dihydroorotate dehydrogenase|Dihydroorotate dehydrogenase]] A (DHODA) ({{PDB|2DOR}}) is an example of a TIM barrel possessing β-sheets and extended loops over the C-terminal end of the β-barrel. DHODA catalyzes the oxidation of dihydroorotate to orotate, which is part of the ''de novo'' uridine 5'-monophosphate (UMP) synthesis pathway. This oxidation is mediated by flavin mononucleotide (FMN). Here, β-sheets and extended loops enclose the active site forming a cavity, while also hosting several catalytic residues.

The ''Methylophilus methylotrophus'' [[Biology:Trimethylamine dehydrogenase|trimethylamine dehydrogenase]] ({{PDB|2TMD}}) TIM barrel is an example of a complete domain insertion. Here, a [[Biology:Rossmann fold|Rossmann fold]] domain is inserted at the C-terminal end of the TIM-barrel. Trimethylamine dehydrogenase catalyzes the conversion of trimethylamine to formaldehyde. This reaction requires both a reduced 6-S-cysteinyl Flavin mononucleotide (FMN) cofactor and a reduced iron-sulphur ([4Fe-4S]<sup>+</sup>) center. FMN is covalently bound within the C-terminal region of the β-barrel. The [4Fe-4S]<sup>+</sup> center is too large to be accommodated within the TIM barrel, and is instead placed in close proximity, 7 Å away, at the interface between the TIM barrel and Rossmann fold domains.

=== 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<ref name=forsyth2007topology>{{cite journal | vauthors = Forsyth WR, Bilsel O, Gu Z, Matthews CR | title = Topology and sequence in the folding of a TIM barrel protein: global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (betaalpha)8 barrel of unknown function from B. subtilis | journal = Journal of Molecular Biology | volume = 372 | issue = 1 | pages = 236–53 | date = September 2007 | pmid = 17619021 | doi = 10.1016/j.jmb.2007.06.018 }}</ref><ref name=carstensen2012conservation>{{cite journal | vauthors = Carstensen L, Sperl JM, Bocola M, List F, Schmid FX, Sterner R | title = Conservation of the folding mechanism between designed primordial (βα)8-barrel proteins and their modern descendant | journal = Journal of the American Chemical Society | volume = 134 | issue = 30 | pages = 12786–91 | date = August 2012 | pmid = 22758610 | doi = 10.1021/ja304951v }}</ref> and 2 designed TIM barrel variants<ref name=carstensen2012conservation /> 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.<ref name=forsyth2007topology/><ref name=carstensen2012conservation /> 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.<ref name=gu2007structural>{{cite journal | vauthors = Gu Z, Rao MK, Forsyth WR, Finke JM, Matthews CR | title = Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Gō model simulation | journal = Journal of Molecular Biology | volume = 374 | issue = 2 | pages = 528–46 | date = November 2007 | pmid = 17942114 | pmc = 2735044 | doi = 10.1016/j.jmb.2007.09.024 }}</ref> Native-centric molecular dynamics simulations recapitulate the experimental results and point the way to testable computational models for complex folding mechanisms.<ref name=halloran2019frustration>{{cite journal | vauthors = Halloran KT, Wang Y, Arora K, Chakravarthy S, Irving TC, Bilsel O, Brooks CL, Matthews CR | display-authors = 6 | title = Frustration and folding of a TIM barrel protein | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 116 | issue = 33 | pages = 16378–16383 | date = August 2019 | pmid = 31346089 | doi = 10.1073/pnas.1900880116 | pmc = 6697809 | bibcode = 2019PNAS..11616378H | doi-access = free }}</ref>

=== 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<ref name="chan2017correlation">{{cite journal | vauthors = Chan YH, Venev SV, Zeldovich KB, Matthews CR | title = Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints | journal = Nature Communications | volume = 8 | pages = 14614 | date = March 2017 | pmid = 28262665 | doi = 10.1038/ncomms14614 | pmc = 5343507 | bibcode = 2017NatCo...814614C | doi-access = free }}</ref> approach and a competition assay<ref name=hietpas2011experimental>{{cite journal | vauthors = Hietpas RT, Jensen JD, Bolon DN | title = Experimental illumination of a fitness landscape | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 19 | pages = 7896–901 | date = May 2011 | pmid = 21464309 | doi = 10.1073/pnas.1016024108 | pmc = 3093508 | doi-access = free }}</ref> 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 [[Biology:C-terminus|C-terminal]] end of the strands tend to contain the [[Biology:Active site|active site]], which is one reason this fold is so common: the residues required to maintain the structure and the residues that effect [[Biology:Enzyme|enzymatic]] catalysis are for the most part distinct subsets:<ref>{{cite journal | vauthors = Ochoa-Leyva A, Soberón X, Sánchez F, Argüello M, Montero-Morán G, Saab-Rincón G | title = Protein design through systematic catalytic loop exchange in the (beta/alpha)8 fold | journal = Journal of Molecular Biology | volume = 387 | issue = 4 | pages = 949–64 | date = April 2009 | pmid = 19233201 | doi = 10.1016/j.jmb.2009.02.022 }}</ref> The linking loops can, in fact, be so long that they contain other protein domains. Recently, it has been demonstrated that catalytic loops can be exchanged between different TIM barrel enzymes as semiautonomous units of functional groups.<ref>{{cite journal | vauthors = Ochoa-Leyva A, Barona-Gómez F, Saab-Rincón G, Verdel-Aranda K, Sánchez F, Soberón X | title = Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability | journal = Journal of Molecular Biology | volume = 411 | issue = 1 | pages = 143–57 | date = August 2011 | pmid = 21635898 | doi = 10.1016/j.jmb.2011.05.027 }}</ref>

== Evolution and origins ==
thumb|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 ''I<sub>BP</sub>'' intermediate state, partially unfolds to reach the ''I<sub>A</sub>'' intermediate state whose conversion to the subsequent ''I<sub>B</sub>'' intermediate state is rate-limiting. The final step is the conversion of ''I<sub>B</sub>'' to the native state, N. The ''I<sub>A</sub>'' and ''I<sub>B</sub>'' 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 <sup>−1</sup>. 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.

thumb|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 [[Biology:Divergent evolution|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.<ref name="farber1990evolution" /><ref name="Farber1995structure2"/> 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.<ref name="branden1991tim2">{{cite journal| vauthors = Brändén CI |date=1991 |title=The TIM barrel—the most frequently occurring folding motif in proteins|journal=Current Opinion in Structural Biology|volume=1|issue=6|pages=978–983|doi=10.1016/0959-440x(91)90094-a}}</ref> Further studies of these phosphate groups, concluding that 12 of 23 [[Biology:Structural Classification of Proteins database|SCOP]] TIM barrel families diverged from a common ancestor.<ref name="copley2000homology">{{cite journal | vauthors = Copley RR, Bork P | title = Homology among (betaalpha)(8) barrels: implications for the evolution of metabolic pathways | journal = Journal of Molecular Biology | volume = 303 | issue = 4 | pages = 627–41 | date = November 2000 | pmid = 11054297 | doi = 10.1006/jmbi.2000.4152 | doi-access = free }}</ref> Similarly there were hints for common ancestry for 17 of the 21 [[CATH database|CATH]] TIM barrel families.<ref name="nagano2002one" /> Based on these reports, it is considered plausible that the majority of TIM barrel proteins evolved from a common ancestor.

=== Origin through gene duplication and domain fusion ===
thumb|300px|'''Model for the evolution of TIM barrels through gene duplication and domain fusion''', as proposed by Lang ''et al''.<ref name="lang2000structural2"/> 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)<sub>4</sub>, (βα)<sub>2</sub>, 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'' [[Chemistry:Histidine#Biosynthesis|histidine biosynthesis]] pathway.<ref name="lang2000structural2"/> 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.<ref name="lang2000structural2"/> 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 [[Biology:Directed evolution|directed evolution]]. Höcker ''et al.'' first fused two C-terminal halves of HisF, yielding HisF-CC. This construct was then stabilized by the insertion of an internal [[Chemistry:Salt bridge (protein and supramolecular)|salt-bridge]], yielding HisF-C*C.<ref name="hocker2004mimicking" /> Further stepwise stabilization and solubilization of HisF-C*C was achieved by optimizing the half-barrel interface, generating HisF-C**C and HisF-C***C, respectively.<ref name="seitz2007stabilisation2"/><ref name="hocker2009high2"/> The crystal structure of HisF-C***C revealed a 2-fold symmetric TIM barrel, validating the possibility of natural domain fusion. Moreover, Höcker created the first chimeric HisAF and HisFA TIM barrels using HisA and HisF half-barrels.<ref name="hocker2004mimicking" /> These experiments led to the proposal of a novel means of diversification and evolution of TIM-barrel enzymes through the exchange of (βα)4 half-barrel domains amongst preexisting TIM barrels. In accordance with this idea, a high catalytic activity on the HisAF construct was established.<ref name="claren2009establishing2"/> Similarly, chimeric  βα<sub>5</sub>-flavodoxin-like fold (CheY)/HisF TIM barrels,<ref name="bharat2008betaalpha2"/><ref name="eisenbeis2012potential2"/> and a perfectly 2-fold symmetric HisF-based TIM barrel<ref name="fortenberry2011exploring2" /><ref name="carstensen2012conservation" /> have also been created.

The existence of 4/8-fold internal symmetry was suggested based on a computational analysis of TIM barrel sequences.<ref name="soding2006hhrep2"/> For example, ''Escherichia coli'' [[Biology:2-dehydro-3-deoxy-phosphogluconate aldolase|KDPG aldolase]]<ref name="wymer2001directed">{{cite journal | vauthors = Wymer N, Buchanan LV, Henderson D, Mehta N, Botting CH, Pocivavsek L, Fierke CA, Toone EJ, Naismith JH | display-authors = 6 | title = Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli | journal = Structure | volume = 9 | issue = 1 | pages = 1–9 | date = January 2001 | pmid = 11342129 | doi = 10.1016/S0969-2126(00)00555-4 | doi-access = free }}</ref> ({{PDB|1FQ0}}) was suggested to possess a distinct 4-fold symmetry, with discernible 8-fold symmetry. The design of a 4-fold symmetric TIM barrel<ref name="huang2016novo2"/> confirmed the possibility of higher orders of internal symmetry in natural TIM barrels, and will be discussed in detail in the next section. No experimental evidence for the existence of 8-fold symmetric TIM barrels has been reported to date.

== ''De novo'' TIM barrel design ==
thumb|300px|'''sTIM-11, the first successful ''de novo'' TIM barrel design.''' The asymmetric (αβ)<sub>2</sub> units are colored distinctly, highlighting the internal 4-fold symmetry.

The TIM barrel fold has been a long-standing target for ''de novo'' [[Biology:Protein design|protein design]]ers. 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.<ref name="borman2015protein">{{cite news |last1=Borman |first1=Stu | name-list-style = vanc |url=https://cen.acs.org/articles/93/i47/Protein-Designers-Roll-Barrel.html |title=Protein designers roll out a barrel |date=2015 |work=Chemical & Engineering News |issue=47 |volume=93 |page=6 }}</ref>

The Octarellin series<ref name="goraj1990synthesis">{{cite journal | vauthors = Goraj K, Renard A, Martial JA | title = Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the alpha/beta-barrel proteins | journal = Protein Engineering | volume = 3 | issue = 4 | pages = 259–66 | date = March 1990 | pmid = 2188263 | doi = 10.1093/protein/3.4.259 }}</ref><ref name="beauregard1991spectroscopic">{{cite journal | vauthors = Beauregard M, Goraj K, Goffin V, Heremans K, Goormaghtigh E, Ruysschaert JM, Martial JA | title = Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the alpha/beta-barrel packing) | journal = Protein Engineering | volume = 4 | issue = 7 | pages = 745–9 | date = October 1991 | pmid = 1798699 | doi = 10.1093/protein/4.7.745 }}</ref><ref name="houbrechts1995second">{{cite journal | vauthors = Houbrechts A, Moreau B, Abagyan R, Mainfroid V, Préaux G, Lamproye A, Poncin A, Goormaghtigh E, Ruysschaert JM, Martial JA | display-authors = 6 | title = Second-generation octarellins: two new de novo (beta/alpha)8 polypeptides designed for investigating the influence of beta-residue packing on the alpha/beta-barrel structure stability | journal = Protein Engineering | volume = 8 | issue = 3 | pages = 249–59 | date = March 1995 | pmid = 7479687 | doi = 10.1093/protein/8.3.249 }}</ref><ref name="offredi2003novo">{{cite journal | vauthors = Offredi F, Dubail F, Kischel P, Sarinski K, Stern AS, Van de Weerdt C, Hoch JC, Prosperi C, François JM, Mayo SL, Martial JA | display-authors = 6 | title = De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure | journal = Journal of Molecular Biology | volume = 325 | issue = 1 | pages = 163–74 | date = January 2003 | pmid = 12473459 | doi = 10.1016/S0022-2836(02)01206-8 | url = https://authors.library.caltech.edu/25336/3/De%20novo%20Backbone%20and%20Sequence%20Design%20of%20an%20Idealized%20alpha-beta-barrel%20Protein-Evidence%20of%20Stable%20Tertiary%20Structure%20%28Offredi%202003%29.pdf }}</ref><ref name="figueroa2013octarellin">{{cite journal | vauthors = Figueroa M, Oliveira N, Lejeune A, Kaufmann KW, Dorr BM, Matagne A, Martial JA, Meiler J, Van de Weerdt C | display-authors = 6 | title = Octarellin VI: using rosetta to design a putative artificial (β/α)8 protein | journal = PLOS ONE | volume = 8 | issue = 8 | pages = e71858 | date = 2013 | pmid = 23977165 | doi = 10.1371/journal.pone.0071858 | pmc = 3747059 | bibcode = 2013PLoSO...871858F | doi-access = free }}</ref> 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 [[Physics:Circular dichroism|circular dichroism]] spectra consistent with αβ proteins and some cooperative folding characteristics, all Octarellin series peptides were insoluble, and had to be resolubilized from [[Biology:Inclusion bodies|inclusion bodies]] for further characterization. Interestingly, Octarellin V.1<ref name="figueroa2016unexpected">{{cite journal | vauthors = Figueroa M, Sleutel M, Vandevenne M, Parvizi G, Attout S, Jacquin O, Vandenameele J, Fischer AW, Damblon C, Goormaghtigh E, Valerio-Lepiniec M, Urvoas A, Durand D, Pardon E, Steyaert J, Minard P, Maes D, Meiler J, Matagne A, Martial JA, Van de Weerdt C | display-authors = 6 | title = The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools | journal = Journal of Structural Biology | volume = 195 | issue = 1 | pages = 19–30 | date = July 2016 | pmid = 27181418 | doi = 10.1016/j.jsb.2016.05.004 | hdl = 2268/199167 | url = https://orbi.uliege.be/handle/2268/199167 }}</ref> displayed a [[Biology:Rossmann fold|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 ([[Physics:Nuclear magnetic resonance|nuclear magnetic resonance]]), and further work to solve their structures could not be pursued.

Proteins of the sTIM series<ref name="huang2016novo2"/> represented the first successful ''de novo'' TIM barrel design.<ref name="nanda2016protein">{{cite journal | vauthors = Nanda V | title = Protein Design: Getting to the bottom of the TIM barrel | journal = Nature Chemical Biology | volume = 12 | issue = 1 | pages = 2–3 | date = January 2016 | pmid = 26678608 | doi = 10.1038/nchembio.1987 }}</ref><ref name="borman2015protein" /> sTIM-11 ({{PDB|5BVL}}) was designed with an internal 4-fold symmetry, to reduce the complexity of computational design using the Rosetta software suite.<ref name="kaufmann2010practically">{{cite journal | vauthors = Kaufmann KW, Lemmon GH, Deluca SL, Sheehan JH, Meiler J | title = Practically useful: what the Rosetta protein modeling suite can do for you | journal = Biochemistry | volume = 49 | issue = 14 | pages = 2987–98 | date = April 2010 | pmid = 20235548 | doi = 10.1021/bi902153g | pmc = 2850155 | doi-access = free }}</ref> Previously-derived first principles<ref name="koga2012principles">{{cite journal | vauthors = Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT, Baker D | title = Principles for designing ideal protein structures | journal = Nature | volume = 491 | issue = 7423 | pages = 222–7 | date = November 2012 | pmid = 23135467 | doi = 10.1038/nature11600 | pmc = 3705962 | bibcode = 2012Natur.491..222K }}</ref> were used to delineate secondary structure topologies and lengths. sTIM-11 proved to be a highly [[Biology:Thermostability|thermostable]], cooperatively folding design that adopted its intended structure.

== See also ==
* [[Biology:Protein folding|Protein folding]]
* [[Biology:Triosephosphate isomerase|Triosephosphate isomerase]]

== References ==
{{Reflist}}

== External links ==
* [http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.d.b.html SCOP list of proteins adopting the TIM barrel fold]
* {{cite web |last1=Babu |first1=M. Madan | name-list-style = vanc |date= 1998 |title=TIM Barrel Analysis |url=http://www.mrc-lmb.cam.ac.uk/genomes/madanm/articles/timanal.htm |website=Center for Biotechnology, Anna University}}

{{Protein tandem repeats}}

{{Protein tertiary structure}}

[[Category:Protein folds]]

[[Category:Protein superfamilies]]


[[Category:Protein domains]]

{{Sourceattribution|TIM barrel}}