The ExPaSy ProtParam tool was used to derive basic information concerning EtnR2 based on its amino acid sequence.
It was calculated that:
•Number of amino acids: 198
•Molecular weight: 21803.5
•Theoretical pI: 4.65
It was also found that EtnR2 has an instability index of 21.05, classifying the protein as stable.
The NCBI BLAST tool was used to conduct a protein BLAST (Basic Local Alignment Search Tool) to identify proteins with close sequence similarity to the EtnR2 sequence. The SmartBLAST feature selects ‘the three best matches in the sequence database together with the two best matches from well-studied reference species, showing phylogenetic relationships based on multiple sequence alignment and conserved protein domains.’
In the case of EtnR1, there were no matches from well-studied reference species. The top matches were all hypothetical Mycobacterium proteins. Figure 1 shows the phylogenetic tree constructed based on sequence similarities and conserved domains. The individual alignments can be found at the bottom of the page.
Figure 1. Phylogenetic tree produced by SmartBLAST with query: hypothetical protein [Mycobacterium chubuense] (accession: WP_014805816.1, GI: 504618714).
Other candidates shown are hypothetical protein [Mycobacterium tusciae] (accession: WP_006247393.1, GI: 493289683), hypothetical protein [Mycobacterium sp. JS623] (accession: WP_015305843.1 GI: 505118741) and hypothetical protein [Mycobacterium rhodesiae] (accession: WP_005148477.1 GI: 491290461).
The BLAST analysis also revealed a predicted MEDS domain (Figure 2). The MEDS domain has been characterised in in hydrocarbon sensing in Methylococcus and is generally fused to a histidine kinase.
Figure 2. Snapshot of BLAST results, showing putative HTH-30 domain at the C-terminal end of EtnR2.
The UCL Bioinformatics PsiPred tool was used to predict the secondary structure of EtnR2 based on its amino acid sequence (Figure 3). The putative helices and sheets were generally predicted with high confidence.
Figure 3. Secondary structure prediction using PsiPred for EtnR2 based on amino acid sequence.
The SWISS-MODEL server [1-4] is an automated service that selects the best matching template to a submitted amino acid sequence, performs the alignment and builds a 3D model. The most important limiting factor of the quality of the model is generally the matching of the query sequence with an appropriate template.
SwissProt threaded EtnR2 onto a selected template protein: flagellar accessory protein FlaH from Methanocaldococcus jannaschii. It is a conserved component of the archaeal flagellum system.
Based on the structure of the template, a homology model was constructed (Figure 4). The predicted structure consisted of a parallel beta sheet surrounded by alpha helices.
Figure 4. Homology model of EtnR2
hypothetical protein [Mycobacterium rhodesiae]
Sequence ID: WP_005148477.1 Length: 197 Number of Matches: 1
Identical Proteins-Identical proteins to WP_005148477.1
Range 1: 1 to 197
hypothetical protein [Mycobacterium tusciae]
Sequence ID: WP_006247393.1 Length: 199 Number of Matches: 1
Range 1: 1 to 199
hypothetical protein [Mycobacterium sp. JS623]
Sequence ID: WP_015305843.1 Length: 199 Number of Matches: 1
Identical Proteins-Identical proteins to WP_015305843.1
Range 1: 1 to 199
1. Marco Biasini, Stefan Bienert, Andrew Waterhouse, Konstantin Arnold, Gabriel Studer, Tobias Schmidt, Florian Kiefer, Tiziano Gallo Cassarino, Martino Bertoni, Lorenza Bordoli, Torsten Schwede. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research; (1 July 2014) 42 (W1): W252-W258; doi: 10.1093/nar/gku340.
2. Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201.
3. Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 37, D387-D392.
4. Guex, N., Peitsch, M.C., Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective.Electrophoresis, 30(S1), S162-S173.