Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008 Sequence and Structure Comparison Studies of Phycocyanin in Spirulina Platensis
Lakshmi, P.T.V. 1 *, Uma Maheswari, S.1 , Karthikeyan, P.P.1 , and Annamalai A.2 1Phytomatics Laboratory, Department of Bioinformatics, Bharathiar University, Coimbatore-46, Tamil Nadu, India, Fax: 0422-2424387; E-mail: [email protected], [email protected] 1Department of Bioinformatics, Aloysius Institute of Computer Sciences, St. Aloysius College, Light House Hill, Mangalore -3, Karnataka, India. E-mail: [email protected] 2Plant Cell and Molecular Biology Laboratory, School of Biotechnology, Karunya University, Coimbatore – 114. Tamil Nadu, India, E. mail: [email protected] *Corresponding author: Lakshmi, P.T.V., E-mail: [email protected]
Received September 01, 2008; Accepted November 10, 2008; Published December 26, 2008
Citation: Lakshmi PTV, Uma MS, Karthikeyan PP, Annamalai A (2008) Sequence and Structure Comparison Studies of Phycocyanin in Spirulina Platensis. J Comput Sci Syst Biol 1: 063-072. doi:10.4172/jcsb.1000005
Copyright: © 2008 Lakshmi PTV, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Crystal structure of Spirulina platensis for Phycocyanin with PDB ID 1GH0 was revealed to contain 24 chains named from 1GH0A to 1GH0X. It was observed that the alternate chains consisted of same sequence however, the odd chains (1GH0A, 1GH0C, 1GH0E… 1GH0W) and even chains (1GH0B, 1GH0D, 1GH0F… 1GH0X) contained 162 and 172 amino acid residues respectively in a similar pattern. Sequence comparison revealed 100 BLAST hits and phylogenetic tree was traced for alternate chains. Similarity percentages of hits were calculated for 1GH0A chain was revealed to have 84 % hits of cyanobacterial sequences, 12 % hits of rhodophyta se- quences, and 4% hits of eugliphida, cyanophora and artificial vector sequences respectively. Similarity percent- ages of hits were calculated for 1GH0B chain was revealed to have 73 % hits of cyanobacterial sequences, 20% hits of rhodophyta sequences, and 5% hits of cryptophyta sequences, and 1% hits of eugliphida and 1% hits of cyanophora sequences respectively. Structure comparisons of these sequences examined by VAST showed resi- dues of alternate entire chains from 1 to 162 and from 1 to 172 residues to contain 1323 structure neighbors. 1628 structure neighbors were found for the phycobilisome domain family which is the major accessory light-1628 harvesting complexes of cyanobacteria and red algae.
Keywords: Spirulina platensis; Phycocyanin; BLAST; Phylogeny; VAST; Structural neighbors
Background
Arthrospira (Spirulina) is an economically important fila- al., 2004; Chen et al., 2006). Spirulina a potential source mentous cyanobacterium. The annual production of the al- of phycocyanin is exploited commercially due to its wide gae is about 10, 000 tons which makes it the largest applications; has stimulatory effect of hematopoiesis (the microalgal cultivation industry in the world (Zhang et al., synthesis of blood), emulating the effect of the hormone 2005). Due to its richness in protein, phycocyanin, essential erythropoietin (Zhang et al., 1994) and regulates the pro- amino acids, polysaccharides, carotenoids, minerals, vita- duction of white blood cells, even when bone marrow stem mins and essential fatty acids has been regarded as an ideal cells are damaged by toxic chemicals or radiation (Evets et bio-resource and has drawn increasing attention in recent al., 1998). Based on these effects, Spirulina is approved decades (Vanshak, 1997; Morist et al., 2001; Kawata et in Russia as “medicine food” for treating radiation sickness. J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 063 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008 Some unique pigments called phycobilins that include phy- mat from the Research Collaboratory for Structural cocyanin and allophycocyanin gives Spirulina a bluish tinge. Bioinformatics -Protein Data Bank. Using Blast Local Align- Among the number of bioactive substances reported in ment Search Tool for Protein (BLAST P) from Geneiouspro Arthrospira, phycocyanin is considered the principal one, 3.7, sequence comparison was performed for the se- with the content up to 10-15% of the dry weight of the alga quences of 1GH0. (Becker, 1994). Geneious Pro is integrated Bioinformatics Software with Phycobilins are attached to proteins forming the phycobi- unique features, native look, and user friendly operability. It lin-protein complex called the phycobiliprotein. Phycobilins can be operated easily because of least training required to are similar in structure to bile pigments such as bilirubin. use the software. It was developed by BIOMATTERS New Since phycocyanin is non toxic and non carcinogenic, it has Zealand in collaboration with University of Auckland and gained importance in synthetic colors, in foods such as al- Oxford University. Common platform combining various coholic drinks, desserts, sweet cake decoration, milk shakes, bioinformatics tools at one place, Geneious Pro is simple to etc., and in cosmetics, thus gaining commercial importance use and effortless to install on any platform. Data consis- especially in pharmaceutical application (Wang et al., 1996; tency across workflows and high interactivity are its ad- Bhat and Madyastha, 2000; Reddy et al., 2003; Subhashini vantages and runs on cross platform (Windows, Linux, Mac et al., 2003). Hence, realizing the importance and a need and Sun Solaris). BLAST P finds regions of local similarity for better identification in terms of enhanced growth, an between sequences. The program compared the query pro- attempt has been made in the present investigation to com- tein sequence 1GH0 to sequence databases and calculated pare the sequence and structure of phycocyanin with PDB the statistical significance of matches and inferred the func- ID 1GH0 (Figure 1) to other closely related and unrelated tional and evolutionary relationships between query protein organisms of algae itself and also with other living organ- sequences 1GH0 with other sequences in the sequence isms using Basic Local Alignment Search Tool for Protein databases. It aided in identifying members of gene families. (BLAST P) from Geneious pro 3.7 and Vector Alignment Search Tool (VAST) respectively. Comparison aids in un- Using Vector Alignment Search Tool of National Center derstanding the system of sequence classification from the for Biotechnology Information (NCBI), structure compari- sequence databases, and structural classification and struc- son was performed, where 3D coordinates on comparison tural neighbors that are available at MMDB/PDB database. provided interesting observation with respect to the align- ments of residues by molecular graphics. However, protein Methodology structure neighbors in Entrez were determined by direct comparison of three dimensional protein structures with the The crystal structure of phycocyanin from Spirulina VAST algorithm. They were compared with more than platensis with PDB ID 1GH0 was retrieved in PDB for-
Figure 1: Crystal Structure of C-Phycocyanin from Spirulina Platensis.
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Figure 2: Phylogenetic tree for similar sequences of the 1GH0A chain.
87,804 domains in Molecular Modeling Data Base (MMDB) Results and from the MMDB (http://www.ncbi.nlm.nih.gov/Struc- Sequence Comparison ture) structure summary pages, retrieved via Entrez, struc- ture neighbors were made available for protein chains and Protein sequence alignment is a way of arranging the pri- individual structural domains were calculated and validated mary sequences of protein to identify regions of similarity according to Hogue et al., (1996). Structural neighbors were that may be a consequence of functional, structural, or evo- presented via 3D molecular graphic images, using the Cn3D lutionary relationships between the sequences (Needleman viewer that is distributed as part of the Entrez client soft- et al., 1970). Aligned sequences of amino acid residues are ware. typically represented as rows within a matrix. Gaps are in- J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 065 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008
Figure 3: Phylogenetic tree for similar sequences of the 1GH0B chain. J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 066 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008 serted between the residues so that residues with identical Phycocyanin had 100 sequence neighbors as per the BLAST or similar characters are aligned in successive columns. P results in total. Similarity percentages of hits were calcu- BLAST P from Geneious pro 3.7 identified a series of short, lated for 1GH0A chain and 1GH0B chain separately by non overlapping subsequences (“words”) in the query se- constructing a phylogram which revealed to have 84 % hits quence that were then matched to candidate database se- of cyanobacterial sequences, 12 % hits of rhodophyta se- quences. quences, and 4% hits of eugliphida, cyanophora and artifi- cial vector sequences respectively (Figure 2). Likewise, the Sequence Neighbors sequence of 1GH0B chain of the crystal structure of Phy- cocyanin again had 100 sequence neighbors as per the The sequence of 1GH0A chain of the crystal structure of
Figure 4: Chain 1GH0A showing residues numbering 1 to 162 and with 57 structure neighbors.
BLAST P results in total. The similarity percentages of hits tures based on their shape and three-dimensional confor- calculated revealed to have 73 % hits of cyanobacterial mation. This process is usually applied to protein tertiary sequences, 20% hits of rhodophyta sequences, and 5% hits structures. In contrast to simple structural superposition, of cryptophyta sequences, and 1% hits of eugliphida and where at least some equivalent residues of the two struc- 1% hits of cyanophora sequences respectively (Figure 3). tures are known, structural alignment requires no a priori knowledge of equivalent positions. Structural alignment is a Structure Comparison valuable method for the comparison of proteins with low Structural alignment is a form of sequence alignment based sequence similarity, where evolutionary relationships between on comparison of shape. These alignments attempt to es- proteins cannot be easily detected by standard sequence tablish equivalences between two or more polymer struc- alignment techniques. J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 067 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008
1 20 40 60 80 100 120 140 160 172 Ch a i n B Ali_Res 3 d D o n + Cha i n B
1KTP B 172
2VML B 172
2UUN B 172
2UUN D 172
2UUL B 172
2UUL D 172
2UUL H 172
2UUN B 172
2UUN X 172
2VJH B 170
2UUN D 1 169
2UUN B 1 169
1KTP B 1 169
2UUN X 1 169
2UUN B 1 169
2UUL H 1 169
2UUL D 1 169
2UUL B 1 169
2VHL B 1 168
2YJH B 1 166
2YML A 161
1KN1 A 159
1JBO A 159
1KN1 B 158
2YML A 1 158
2YJT A 158
2YJT A 1 158
2YJT B 157
2UUN A 157
2UUL A 157
1BBD A 157
2YJH A 157
2UU C 157
2UUL C 157
1KN1 A 1 156
2UUN A 1 156
2UUL A 1 155
1JBO A 1 155
2UUL C 1 155
1KN1 B 1 154
2VJT B 1 154
2YJH A 1 153
1BBD A 1 151
1XGO D 140
1YKU A 120
2BNL C 118
2OR4 H 117
1GVH A 1 106
1UC3 A 91
1HY6 A 1 38 Figure 5: Chain 1GH0B showing residues numbering 1 to 172 and with 53 structure neighbors.
J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 068 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008 Structure Neighbors 1323 structural neighbors.
In the present study, the crystal structure of Phycocyanin In these 24 chains the alternate chains with residue rang- had 1323 structure neighbors as indicated by VAST analy- ing from 36 to162 and from 36 to 172 were found to be sis (Wang et al., 2001). Each alternate chain represented domain coding and were revealed to be phycobilisome pro- with 57 and 53 structural neighbors of both 1GH0A and tein family with 1628 structure neighbors. Phycobilisomes 1GH0B are represented in figure 4 and figure 5 respec- are the major accessory light-harvesting complexes of tively. Interesting, similar fashion was observed for the re- cyanobacteria and red algae. Phycobilisomes are mainly maining chains from 1GH0C to 1GH0X, together making to composed of phycobiliproteins (such as allophycocyanin,
1 20 40 60 80 100 120 140 160 Ali_Res Chain A 3d Don.
1JBO A 159
1JBO A 1 159
2UUN A 159
2UUN A 1 159
2UUL A 159
2UUL A 1 159
2UUL C 159
2UUL C 1 159
2YML A 157
2YML A 1 157
2YJT A 157
2YJT A 1 157
2UUL H 156
2UUM B 156
1KN1 A 156
1KN1 A 1 156
1BBD A 156
1BBD A 1 156
2YJH A 156
2YJH A 1 156
2UUM B 1 155
2UUN D 155
2YJT B 155
2YJT B 1 155
2UUL B 154
2UUL B 1 154
2UUL H 1 154
2UUL D 154 2UUL D 1 154
2UUM X 154
2UUM X 1 154
2YNL B 1 154
2YJH B 154
2YJH B 1 154
1KTP B 153
1KTP B 1 153
2UUN D 1 153
2UUN B 153
2UUN B 1 153
2YNL B 153
1KN1 B 147
1KN1 B 1 147
1XGO D 121
2DC3 A 117
1OR4 A 115
1HLB A 114
1X3K A 114
1OJ6 B 111
1UC3 A 111
1YKU A 110
2LHB A 109
2BNL C 108
1A87 A 2 107
1A87 A 106
1COL A 106
1CII A 3 97
2YEB A 97
2ZFO D 87
1KR7 A 86
2I88 A 80 Figure 6: Domains of chain 1GH0A with residues from 36 to 162 having 73 structure neighbors. J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 069 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008
1 20 40 60 80 100 120 140 160 172 Chai n B Ali_Res 3d Don. 1
1KTP B 169
1KTP B 1 169
2UUN B 169
2UUN B 1 169
2UUN D 169
2UUN D 1 169
2UUL D 169
2UUL D 1 169
2UUL H 169
2UUL H 1 169
2UUN B 169
2UUN B 1 169
2UUN X 169
2UUN X 1 169
2YJT A 155
2UUL A 155
2UUL A 1 155
2YJT B 154
2YJT B 1 154
2YJH A 154
1KN1 B 1 154
2UUN A 1 154
1BBD A 152
2YJH A 1 151
1XGO D 139
1YKU A 120
1OR4 A 117
2BNL C 115
1COL A 104
1GVH A 1 95
2YEB A 93
1UC3 A 91
1KR7 A 79
1PDZ A 1 52
1TE6 A 1 52
2ES9 A 47
1WY6 A 1 38 Figure 7: Domains of chain 1GH0B with residues ranging from 36 to 172 with 60 structure neighbors. phycocyanin and phycoerythrin) together with linker also known as polypeptides consist of a sequence of 20 polypeptides. Figure 6 and figure 7 represents the two iden- different L-á-aminoα acids, also referred to as residues tified domains of chain 1GH0A and 1GH0B respectively. (Pauling et al., 1951). However, advancement in this scien- The domain of chain 1GH0A coding with amino acids ranged tific world led to the concept of structural biology that em- from 36 to 162 and that of chain 1GH0B with residues ranged ploys techniques such as X-ray crystallography or NMR from 36 to 172 respectively. Like wise domains for the chains spectroscopy to determine the structure of proteins which named 1GH0C to 1GH0X altogether made 1628 structure are determined and stored in Protein Data Bank (PDB). neighbors in total. The crystal structure of Phycocyanin retrieved from PDB Discussion contained 24 chains and were named from 1GH0A to 1GH0X of which, every alternate chains had the same sequence for Proteins are an important class of biological macromol- the odd chains (1GH0A, 1GH0C, 1GH0W) and even chains ecules present in all biological organisms, made up of such (1GH0B, 1GH0D, 1GH0X) respectively. Sequence neigh- elements as carbon, hydrogen, nitrogen, oxygen, and sulfur. bors numbering 100 were found and phylogenetic tree was All proteins are polymers of amino acids. The polymers, traced for the alternate chains of 1GH0 structure of phyco-
J Comput Sci Syst Biol Volume 1 : 063-072 (2008) - 070 ISSN:0974-7230 JCSB, an open access journal Journal of Computer Science & Systems Biology - Open Access Research Article JCSB/Vol.1 2008 cyanin of Spirulina platensis. Similarity percentages of hits References were calculated for 1GH0A chain was revealed to have 84 % hits of cyanobacterial sequences, 12 % hits of rhodophyta 1. Becker EW (1994) Microalgae: biotechnology and mi- sequences, and 4% hits of eugliphida, cyanophora and arti- cro-biology. Cambridge University Press London. » CrossRef ficial vector sequences respectively. Similarity percentages » Google Scholar of hits were calculated for 1GH0B chain was revealed to 2. 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