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. 2013 Aug 12;8(8):e70582.
doi: 10.1371/journal.pone.0070582. eCollection 2013.

Alternative Splice Variants in TIM Barrel Proteins From Human Genome Correlate With the Structural and Evolutionary Modularity of This Versatile Protein Fold

Free PMC article

Alternative Splice Variants in TIM Barrel Proteins From Human Genome Correlate With the Structural and Evolutionary Modularity of This Versatile Protein Fold

Adrián Ochoa-Leyva et al. PLoS One. .
Free PMC article


After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)2, (βα)4, (βα)5, (βα)6, and (βα)7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Pipeline of bioinformatics analysis to identify the (βα)8 barrel proteins with splice variants in the human genome.
Summary of our bioinformatics data flow to extract the 135 experimentally confirmed (βα)8 barrel splice variants in the human genome.
Figure 2
Figure 2. Subdomain distribution of 67 splice variants found in the (βα)8 barrel proteins from the human genome.
The different (βα)8 barrel subdomains present in the splice variants are illustrated in a). The typical secondary-structure composition of the canonical (βα)8 barrel consists of eight repeats of βα modules. The secondary-structure composition of different (βα)8 barrel subdomains is described in A. The number of splice variants for each subdomain category is as follows: (βα)1 = 1, (βα)2 = 3, (βα)3 = 3, (βα)4 = 8, (βα)5 = 6, (βα)6 = 10, (βα)7 = 23, others  = 13. The subgroup of the 13 splice variants that cause structural modifications to the (βα)8 barrel proteins without altering their overall fold structure are illustrated in B. The number of splice variants for each category is as follows: β-strand substitution  = 5, α-helix deletion  = 3, insertion and deletion in loops  = 3, (βα) substitution  = 1, α-helix substitution  = 1. All the illustrated data were taken from Table S5.
Figure 3
Figure 3. Sequence analysis under selective pressure at variable positions in chimera enzymes.
Amino acids are represented by the one code letter. The histograms represent the relative frequencies of the selected versus unselected libraries. The red colored histograms (*) represent those amino acids at the variable positions that had a frequency either significantly higher (2 standard deviations) or lower than expected by chance. The sequence analysis for the α-helix 3 and β-strand 7 from MetR swapped into the TrpF scaffold are shown in A and B, respectively. The variable positions are represented by the NNS codon. The three-dimensional structure of the E. coli enzyme (PDB: 1PII) was used to identify the variable positions. The amino acid numbering of TrpF is according to gene reported in .
Figure 4
Figure 4. Structural analysis of variant Beta_1.
The variant Beta_1 has a leucine residue in the N-terminal variable position and a glycine residue in the C-terminal variable position. A. Far-UV CD spectra. B. Thermal unfolding curves. C. Analytical gel-filtration chromatograms. A. The variant Beta1 showed only slight structural changes at the secondary structure level with respect to the wild-type enzyme. B. The variant Beta_1 (brown squares) has a Tm that is 5 degrees higher than the wt-TrpF enzyme (green triangles). C. The variant Beta_1 has an increase in the dimer population relative to the wild-type TrpF.
Figure 5
Figure 5. A model of structural assembly of novel (βα)8 barrels by splice variants.
According to the experimental evidence of the existence of soluble and stable barrel subdomains in many different (βα)8 barrels, we suggest a process of subdomain assembly through genomic evolution which may result in multiple lineages of novel (βα)8 barrels in the human genome. In addition, the splice variants found in the human genome possibly form homo-dimers, heterodimers, or three-quarter barrel + quarter-barrel complexes to complete the (βα)8 barrel structure. The ribbon diagram shows a representation of the canonical (βα)8 barrel structure, and the different colors correspond to subdomains of different proteins that can be reassembled in the complete barrel (center). This model provides additional support for the proposed models on the origin of the (βα)8 barrel fold through the assembly of smaller subdomains. The 3D structure of PDB 1AW1 was used to illustrate the model.

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This work was supported by National Institute of Genomic Medicine grants 08/2012/I and 07/2012/E. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.