Supporting InformationBarua et al. 10.1073/pnas.1101221108SI Materials and MethodsData Acquisition. The cDNA sequences of tropomyosin (Tm)isoforms were collected from online databases such as NationalCenter for Biotechnology Information, Ensembl GenomeBrowser, and University of California, Santa Cruz (UCSC)Genome Browser through keyword searches for “tropomyosin”or BLAST searches with protein sequences as the query. Withfew exceptions all selected species have annotated genomes.Our goal was to maximize the depth of the phylogenetictree. The species that were included in our study are: Homosapiens, Macaca mulatta, Rattus norvegicus, Monodelphis domes-tica (Mammalia); Gallus gallus (Aves); Xenopus laevis, Xenopustropicalis (Amphibia); Danio rerio, Takifugu rubripes, Tetraodonnigroviridis (Teleostei); Petromyzon marinus (Agnatha); Cionaintestinalis (Urochordata); Branchiostoma floridae (Cephalochor-data); Strongylocentrotus purpuratus (Echinodermata); Sacco-glossus kowalevskii (Hemichordata); Drosophila melanogaster,Bombyx mori, Homarus americanus, Limulus polyphemus, Derma-tophagoides pteronyssinus (Arthropoda); Caenorhabditis elegans(Nematoda); Mizuhopecten yessoensis, Biomphalaria glabrata(Mollusca); Schistosoma mansoni (Platyhelminthes); and Nema-tostella vectensis, Hydra magnipapillata (Cnidaria) (Table S1).BLAST searches also identified fungal tropomyosin sequences,but we did not include them in the present phylogenetic analysis.However, we did not find any candidate sequences for plant,protist, or Porifera species.

A large number of Tm cDNA sequences from the databasesare partial coding sequences, are not correctly annotated, orare derived from predictions by automated annotation programs.A careful evaluation of all sequences was carried out throughmanual inspection and by applying our knowledge of the biologyand structure of Tm to filter out or edit the sequences that wereunlikely to be Tms. For example, sequences of some transcriptswere unlikely to exist based on comparisons to Tms of closelyrelated organisms including the presence of internal helix-break-ing residues such as Pro known not to be present in vertebrateTms. We have only included those sequences for which EST ormRNA evidence was available and/or if the sequences were simi-lar to the sequences of Tm isoforms present in closely relatedorganisms. The low sequence complexity resulting from the re-dundant heptad repeats diagnostic of coiled-coil sequences madesearching for phylogenetically distant sequences a challenge. Weincluded sequences in which the coiled coil began with the initi-ating Met, and proteins that were approximately 284 residues orshorter. The shortest full-length sequence included in our analysisencodes a 242-residue protein. In well-annotated invertebrategenes with proline-rich terminal exons, as in D. melanogasterTPMI, the proline-rich exon was not included.

Because most invertebrate Tm sequences are very differentfrom the vertebrate sequences, identifying these as tropomyosinsrequired a more detailed analysis. Exon numbers are not strictlyconserved between invertebrate and vertebrate genes (1, 2).Some exons are split and others are fused relative to the verte-brate exon structure but the exon junctions, when present, areconserved. For example, in the sea urchin, exon 4 (39 aa), asillustrated in Fig. S1, actually consists of two exons, 4 (15 aa) and4′ (24 aa), and exons 6 (25 aa) and 7 (21 aa) in the figure areactually one exon in the sea urchin (46 aa). We only included in-vertebrate sequences in which the exon junctions presentcorrespond to those in the vertebrates irrespective of the exonnumber. All exons were named based on their nomenclature

in the vertebrate genes. Only the coding regions of genes wereincluded in the analysis.

Throughout we were parsimonious in our selection ofsequences. Based on the criteria outlined above we identified Tmsequences from Mnemiopsis leidyi (Ctenophora), and Monosigabrevicollis (Choanoflagellida) (Table S1) but we did not includethem in our dataset because there were insufficient exons torelate the sequences to invertebrate exons, and the sequenceswere too different from others Tms for alignment. Additionalsequences identified as tropomyosins were eliminated fromour final tree, including sequences that are highly divergent intrees that were published after we completed our analysis (1).For example, we included 3 of 6 predicted genes in C. intestinalis;1 of 7 predicted genes inN. vectensis; and 1 of 6 predicted genes inH. magnipapillata. We did not include sequences from severaladditional invertebrate species because they were incomplete, orwe were not confident about our ability to validate them as Tms.

Sequence Alignments. Because each Tm gene can produce severalisoforms through alternative splicing of exons and use of alter-nate promoters, the sequences of all available isoforms werecollected. The cDNA, EST, or genomic sequences were editedto obtain the coding regions. All sequences expressed by the samegene were manually aligned in SEAVIEW (3) so that all theexons, including the alternatively spliced exons (when present),were arranged in the following order: 1a-2a-2b-1b-3-4-5-6a-6b-7-8-9a-9b-9c-9d, which is the order of exons in vertebrate Tmgenes (Fig. S1). The lack of complexity of the coiled coil sequenceresulted in unsatisfactory results using automated alignmentprograms. The final sequence for each gene was created by usingthe “consensus sequence” option of SEAVIEW based on thesequence alignments of all isoforms to give the coding sequenceof the genes with the exons in the order found in the genome.

Phylogenetic analyses often align the coding regions withoutrespect to alternative exons (1). In doing so, from the point ofview of an evolutionary analysis, the constitutive exons are over-represented and the alternate exons are compiled, whereas theyare encoded by different sites in the gene. We arrived at theapproach used in our study following discussions with Facultyof the 2008 Workshop on Molecular Evolution at the MBL,Woods Hole, MA, in which S.E.H.-D. was a student.

The invertebrate tropomyosins either have no alternativelyspliced exons or have alternative splicing of exons that areconstitutive in the vertebrates (1, 2), introducing an additionalchallenge in the alignment of the vertebrate and invertebratesequences. Therefore, alignment of the invertebrate gene se-quences with the vertebrate sequences was based on exon treesthat were constructed separately for each exon. For example, allalternative exon 9 sequences from vertebrates and invertebrateswere aligned and a phylogenetic tree was constructed. The exontrees were constructed using MrBayes using the same parametersthat were used for constructing the trees with the complete genesequences. The resulting exon 9 tree indicated that the duplica-tions of exon 9, designated as 9a–d in vertebrates and 9A–E ininvertebrates, originated independently in the vertebrate and in-vertebrate lineages, in agreement with another recently publishedanalysis (1). However, exon 9a of vertebrates was closest to exon9B of invertebrates in phylogeny. Therefore exons 9a and 9B werealigned with each other, and the alternate exons 9b–d of thevertebrates were aligned separately from the alternate exons 9A,C–E of the invertebrates. This gave rise to the following order ofexon 9s in the total dataset: 9A-9a/9B-9b-9c-9d-9C-9D-9E, where

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9a–d are alternate forms of vertebrate exons and 9A–E are alter-nate forms of invertebrate exons (Fig. S1). The exons have beennamed a–d or A–E in the order in which they are present in theTm genes based on genomic data. A similar analysis was carriedout for all the other exons, 1–8. The order of alignment of thevertebrate and invertebrate exons based on this analysis is shownin Fig. S1 and the complete sequence alignments are shown inDataset S1. For each exon tree the sequences were identifiedand aligned according to the length of the vertebrate exons, eventhough in certain cases, as noted above, they are fused or splitin the invertebrates. Our “genome-based” sequence alignmentdiffers from that of Irimia et al. (1) who aligned the sequenceswithout separating alternate exons. Because the exon duplica-tions occurred independently, after divergence of the vertebrateand invertebrate lineages, there was little impact on the calcu-lated trees. Their method of alignment, though useful for theirinterest in internal and external paralogy, is not valid for the evo-lutionary analysis that we carried out here, as explained above.

Evolutionary Analysis. Phylogenetic trees were constructed fromthe Tm gene sequences using two different approaches forphylogenetic analyses: maximum-likelihood (GARLI) (4) andBayesian (MrBayes) (5). The evolutionary model was JTT+G+F,determined using Prottest (6). GARLI 1.0 was used for the max-imum-likelihood analysis (4). Branch support was obtained with500 bootstrap replicates with 2 search replicates per bootstrap.The 50% majority consensus tree was obtained in PAUP* 4.0(7). The Bayesian analysis was done using MrBayes v3.2 (5) withtwo independent runs of 4 simultaneous chains for 1,000,000 gen-erations and a sample frequency of 100. The average standarddeviation of split frequencies was <0.01 at the end of the runs.The consensus tree was constructed after discarding 25% of thesamples. Branch support was obtained as posterior probabilities.

PAML version 4.1 (8) was used to calculate the substitutionrates (ω) at individual codon sites using variable site codon mod-els M3 and M7 with 4 rate categories (K ¼ 4) under CODEMLwith the the GARLI and MrBayes trees. CODEML was firstrun under model M0 (no variation in ω among sites) with codonsubstitution model F61. The branch lengths obtained under mod-el M0 were used for the analysis with variable site models M3 andM7 to avoid prohibitive computational times. The ω values wereobtained as the posterior mean, where ω ¼ ω0�PP0þ ω1�PP1þω2�PP2 and so on for each ω category, where PP0, PP1 etc. arethe posterior probabilities of categories ω0, ω1 etc, respectively.The average ω value for each site was calculated from the poster-ior means of the ω values obtained using the two models andthe two trees (Table S2). In all cases ω < 1, meaning there is noneutral or positive selection for any codon.

DNA Construction and Protein Purification.Mutations were made inrat striated α-tropomyosin cDNA (encoded by the TPM1 gene;exons 1a-2b-3-4-5-6b-7-8-9a) with an Ala-Ser extension at theN-terminus, cloned in pET11d for expression in Escherichia coli.Recombinant AS-αTm binds well to actin in the absence oftroponin, unlike recombinant unacetylated-αTm (9). Mutationswere made using oligonucleotides and their reverse complementsusing two-stage PCR as previously described (10). The mutationswere verified by sequencing of the DNA at the DNACore Facilityat the Robert Wood Johnson Medical School (RWJMS) andGenewiz. Mutants were expressed in E. coli. BL21(DE3) cellsand purified as previously described (10). Actin was purified fromacetone powder of chicken pectoral skeletal muscle actin (11).Tm and actin concentrations were determined by measuringthe difference spectrum of tyrosine (10). The molecular weightsof the purified proteins were verified by electrospray massspectrometry at the Keck Biotechnology Resource Lab, YaleUniversity (Table S3).

Actin Binding Assays. Tm (0.1–8 μM) was combined with 5 μMF-actin and cosedimented at 20 °C in 200 mM NaCl, 10 mMTris-HCl, pH 7.5, 2 mM MgCl2, and 0.5 mM DTT (12). Thepellets and supernatants were analyzed by SDS-PAGE, stainedwith Coomassie blue, and scanned and analyzed using Image-Scanner III densitometer with Labscan 6.0 and ImageQuantTL 7.0 image analysis softwares. The free Tm in the supernatantswas calculated from standard curves for WT-Tm. The bindingconstant Kapp and the Hill coefficient (αH) were determinedby fitting the experimental data to the Hill equation (10) usingSigmaPlot:

ν ¼ ðn½Tm�αHKαHappÞ∕ð1þ ½Tm�αHKαH

appÞ;

where ν ¼ fraction maximal Tm binding to actin, n ¼ maximalTm bound, and ½Tm� ¼ ½Tm�free. The Tm∶actin ratio was normal-ized to 1 by dividing the Tm∶actin ratio obtained from densito-metry by the Tm∶actin ratio observed at saturation.

Circular Dichroism Measurements. Thermal stability measurementswere made by following the ellipticity of 1.5 μM Tm at 222 nm asa function of temperature in 0.5 M NaCl, 10 mM sodium phos-phate, pH 7.5, 1 mMEDTA and 1 mMDTT in an Aviv model 400spectropolarimeter at the RWJMS CD facility. The observedmelting temperature (TM) is defined as the temperature at whichthe ellipticity at 222 nm, normalized to a scale of 0 to 1, is equal to0.5 (13).

Differential Scanning Calorimetry Measurements. DSC experimentswere performed on a Model 6100 Nano II differential scanningcalorimeter with 0.3 mL cells at a scanning rate of 1°C∕minute,and pressure of 2 atm. 15 μM Tm was mixed with phalloidin(Sigma) (36 μM) stabilized F-actin (24 μM) in 100 mM NaCl,10 mM Hepes, pH 7, 2 mM MgCl2, and 1 mM DTT. In scan1, the sample was heated from 0–70 °C and cooled. This curvecorresponds to the unfolding of actin-bound Tm (14, 15). In scan2, the sample was heated from 0–90°C leading to irreversibledenaturation of F-actin and cooled. In scan 3, the sample washeated from 0–70°C. This curve corresponds to the unfoldingof Tm in the absence of actin. Because thermal unfolding ofTm is reversible up to 70 °C, scans 1 and 2 are identical, andour analysis was based on scans 2 and 3. The data was analyzedusing CpCalc and Origin 5.0 (MicroCal) software. The TM valueswere obtained by deconvolution of the heating curves using anon-two state model in Origin 5.0. A molecular weight of66 kDa was used for coiled coil Tm dimers.

Construction of Tm-Actin Model. A working model for Tm–actininteraction was constructed with a 6.6 Å resolution structureof F-actin (16) and 2–3 Å resolution structures of Tm fragments(17–20). The azimuthal and axial alignments were based on aprevious model proposed by Brown et al. (18). The azimuthalposition in their model coincided with that of the Ca2þ-activatedstate, and the axial position was determined by matching anapolar patch in period 5 (P5) (residues 169–172) with the mostprominent apolar surface on actin (residues 329–333 in subdo-main 3). Based on the contacts observed in that model, we dockedV170, E181, and E177 of Tm near P333, K328, and K326 of actin,respectively. Based on the assumption that the target sites onactin are the same in all periods, alignments for P1–P4, andP6, P7 were carried out by docking the hydrophobic and acidicresidues corresponding to V170, E177, and E181 near the actinresidues P333, K328 and K326. The radial distance between tro-pomyosin and F-actin is not defined in our model. The model wasconstructed using the University of California, San Francisco(UCSF) Chimera package from the Resource for Biocomputing,Visualization, and Informatics at the UCSF [supported byNational Institutes of Health (NIH) P41 RR001081] (21).

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SI Results.Evolutionary Analysis. The consensus trees from MrBayes andGARLI showed close relationships between the TPM1 andTPM3 genes, and the TPM2 and TPM4 genes of the vertebratesconsistent with an earlier phylogenetic study of a smaller datasetincluding only exons 3-4-5-6b-7-8 of vertebrate genes (22). It waspostulated in that study that the four TPM genes of the verte-brates arose through gene duplications of a single ancestralTPM gene during early chordate evolution. This is consistentwith the theory that two whole genome duplication events haveoccurred in the lineage leading to vertebrates (23). Therefore,TPM gene duplications arose in the vertebrate and invertebratelineages independent of each other.

It has been suggested that Tm originated by repeated duplica-tion of an ancestral 21 amino acid sequence (24). This is evidentin the repetitive structure of the Tm molecule that has beenhighly conserved in all eukaryotic organisms. Our trees show thatexon duplications leading to alternatively spliced exons withinTPM genes took place independently and on multiple occasionsin the vertebrate and invertebrate lineages, in agreement withIrimia et al. (1). In vertebrates, all TPM genes have alternativeexpression of exons 1 (1a/b), 6 (a/b) and 9 (a-d); exon 2 (a/b) is

alternatively spliced only in the TPM1 gene. In invertebrates, theexons that are alternatively spliced differ between differentspecies. For example, exons 3 (A/B), 4 (A–C) and 9 (B–D) arealternatively spliced in the C. elegans TPM gene, whereas exons4 (A/C), 5 (A/B), 6 (A/B), 7 (A/B), 8 (A/B) and 9 (A/B) are alter-natively spliced in the S. mansoni TPMI gene.

The echinoderms and hemichordates are known to be sisterphyla to the chordates (25), but in our trees they branch outwith the cnidarians indicating a closer relationship to the cnidar-ians than to the chordates. The long branch lengths observed forStr.pur_TPMII and Hyd.mag_TPM (Fig. 1A) at the base of thetree may cause a “long branch attraction artifact” in the tree.However, a tree constructed without these two sequences pro-duced the same tree topology.

There is a possibility that the deep divergences represented byour sample will saturate synonymous substitutions, which wouldimpact the estimates of ω. In that case, ω will primarily providea measure of amino acid variability. However, that was not a con-cern for our analysis, because the ω values were used to generatehypotheses concerning protein structure and function that wereexperimentally tested through mutagenesis studies.

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9. Monteiro PB, Lataro RC, Ferro JA, Reinach FC (1994) Functional alpha-tropomyosinproduced in Escherichia coli. A dipeptide extension can substitute the amino-terminalacetyl group. J Biol Chem 269:10461–10466.

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sin molecules: Implications for actin binding and regulation. J Mol Biol 364:80–96.

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Fig. S1. Exon organization and alignment of the vertebrate and invertebrate TPM genes used for our phylogenetic analysis. This representation relatesthe invertebrate exons to the vertebrates but does not illustrate the variable exon structure of the invertebrates.

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Fig. S2. Conserved residues of tropomyosin based on ω values obtained for the dataset of metazoan sequences. (A) Average ω values of a 284-residueTm sequence with exons 1a-2b-3-4-5-6b-7-8-9a. The black bar represents ω ¼ 0.015; (B) conserved residues with ω ≤ 0.015 at heptad repeat positions a–gshown in the striated muscle αTm structure (exons 1a-2b-3-4-5-6b-7-8-9a) (1C1G) (1). Conserved residues (ω ≤ 0.015) where mutations cause skeletal and car-diomyopathies are shown in black.1 Whitby FG, Phillips GN Jr. (2000) Crystal structure of tropomyosin at 7 Å resolution. Proteins 38:49–59.

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Fig. S3. Actin affinity of tropomyosin mutants. Tropomyosin (0.1–8 μM) was combined with 5 μM F-actin and sedimented at 20 °C in 200 mM NaCl, 10 mMTris-HCl (pH 7.5), 2 mM MgCl2, and 0.5 mM DTT. Stoichiometric binding of one Tm per seven actins is represented by fraction maximal binding of 1. The datafor each mutant and WTwas obtained from two to four independent experiments. The Kapp values are reported in Table 1. Symbols: (●) WT; A. (▴) P3-1-EE,(Δ) P3-2-EKDE; B. (▪) P4-1-RKVE, (□) P4-2-EEEE, (○) P4-null-QE; and C. (♦) P5-2-EKNK, (+) P6-1-K, (◊) P6-2-KEE.

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Fig. S4. Thermal stability of tropomyosin mutants. Fraction folded as measured by the relative ellipticity at 222 nm as a function of temperature. The Tmconcentration was 1.5 μM. The fraction folded is relative to the mean residue ellipticity at 0 °C where the proteins were fully folded. The TM values are reportedin Table 1. Symbols: (●) WT; A. (▴) P3-1-EE, (Δ) P3-2-EKDE; B. (▪) P4-1-RKVE, (□) P4-2-EEEE, (○) P4-null-QE; and C. (♦) P5-2-EKNK, (+) P6-1-K, (◊) P6-2-KEE.

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Fig. S5. DSC scans of tropoomyosin mutants in the presence and absence of F-actin as described in SI Text. Scan 2 (Tmwith F-actin—black solid lines) and scan 3(post-F-actin denaturation Tm—gray dotted lines) are shown. (A) WT; B. P2-1-KDDE; C. P2-null-KKD; D. P4-1-RKV; E. P4-null-QEI; F. P5-1-REEE. The TM1 and TM2

transitions in scans 2 and 3 correspond to the unfolding of the C-terminal and N-terminal regions of free Tm, respectively. The C-terminal region of Tmis stabilized on binding actin and the actin-induced transition in scan 2 (TM3) originates mainly from the unfolding of the stabilized C-terminal regionaccompanied by cooperative dissociation of Tm from actin

Other Supporting Information FilesTable S1 (DOC)Table S2 (DOC)

Table S3 (DOC)Dataset S1

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