A. Sequence and structural analysis of α-crystallin proteins
The 20 amino acid sequences of α- crystallin proteins of 10 habitat-specific fish had been retrieved from NCBI database for our study. For comparison, α-crystallin from Bos taurus was predicted and found to be the closest homologue for the crystallins of the ten fish.
The secondary structure of all the 20 amino acid sequences of α-crystallin protein were predicted using tools like GORIV, SOPMA,PSIPRED listed in ExPASy proteomics server. The 2D structure tools have shown α-helices, β-pleated sheets and coils in all 20 sequences at respective places (Fig. 1, supplementary table 1). The 2D structures of α- crystallin of the sequences have been presented in the form of PSIPRED (Fig. 2I-VI).
Table 1 The number of 2D structures predicted by secondary structure prediction tools.
(H:Alpha Helix, S: Beta-pleated Sheets, C: Coils)
The 3D structures of α-crystallin protein sequences of the five saltwater fish species (Fig. 3–5) revealed an overall structural similarity with higher Z scores, indicating their favourable states. The GMQE values of αB- crystallins of saltwater fish species are generally higher than that of freshwater fish species, thereby indicating that saltwater fish species have favourable states and higher reliabilities, compared to the freshwater species. An exception is sheepshead minnow αB crystallin(2ygd.1.A) which possesses a lower GMQE as compared to zebrafish αB crystallin(2ygd). The αA crystallins of some species possess similar GMQE values for instance, αA crystallins of zebrafish(2ygd) and sheepshead minnow, Green Swordtail with Damselfish(6t1r), Atlantic Salmon with Zigzag Eel. The Ramachandran Plots for the species (Fig. 3–5) showed that all the residues were mostly present in the favourable/allowed regions.
II. Physiochemical Characterisation
The various physiochemical parameters of the α-crystallin proteins of 10 fish species are shown in Fig 6 (supplementary table 2). Some proteins, such as αB- crystallin of two freshwater fish Striped Bass, Atlantic Salmon possess instability indices of 36.22 and 37, 53 respectively, indicating their stabilities. On the other hand, αA-crystallin of saltwater fish Zigzag eel is found to be a stable protein with an instability index of 39.89.
Table 2 Physicochemical parameters of α-crystallin of Bos taurus and the ten habitat-specific fish, computed from Expasy ProtParam.
|
Number of amino acids
|
Molecular weight
|
Theoretical p.I.
|
Total number of negatively charged residues (Asp + Glu)
|
Total number of positively charged residues (Arg + Lys)
|
Extinction Coefficients
|
Instability Index
|
Aliphatic Index
|
Grand average of hydropathicity (GRAVY)
|
Bos taurus(αA)
|
173
|
19790.14
|
5.78
|
25
|
20
|
14440
|
57.45(unstable)
|
72.08
|
-0.494
|
Bos taurus(αB)
|
175
|
20036.79
|
6.76
|
25
|
24
|
13980
|
50.92(unstable)
|
77.43
|
-0.507
|
Freshwater Species
|
|
|
|
|
|
|
|
|
|
Danio rerio(αA)
|
173
|
19713.18
|
5.84
|
25
|
21
|
17545
|
41.16(unstable)
|
66.42
|
-0.643
|
Danio rerio(αB)
|
168
|
19977.59
|
5.77
|
25
|
20
|
33920
|
48.40(unstable)
|
70.18
|
-0.561
|
Bagarius yarreli (αA)
|
275
|
30033.23
|
5.21
|
41
|
29
|
12170
|
46.27(unstable)
|
67.02
|
-0.543
|
Bagarius yarreli(αB)
|
197
|
22017.40
|
4.71
|
34
|
25
|
7115
|
64.65(unstable)
|
70.71
|
-0.561
|
Xiphophorus hellerii(αA)
|
176
|
19890.08
|
5.84
|
25
|
21
|
17420
|
40.37(unstable)
|
69.26
|
-0.585
|
Xiphophorus hellerii(αB)
|
164
|
19200.75
|
5.49
|
23
|
18
|
44585
|
42.39(unstable)
|
66.52
|
-0.484
|
Morone saxatilis(αA)
|
176
|
19912.96
|
5.70
|
25
|
21
|
17420
|
49.51(unstable)
|
61.48
|
-0.634
|
Morone saxatilis(αB)
|
164
|
19255.78
|
6.06
|
24
|
21
|
45950
|
36.22(stable)
|
75.43
|
-0.552
|
Salmo salar(αA)
|
177
|
20045.28
|
5.69
|
25
|
21
|
17545
|
48.34(unstable)
|
58.93
|
-0.605
|
Salmo salar(αB)
|
149
|
16300.58
|
5.62
|
18
|
13
|
8605
|
37.53(stable)
|
80.40
|
-0.219
|
Saltwater Species
|
Number of amino acids
|
Molecular weight
|
Theoretical p.I.
|
Total number of negatively charged residues (Asp + Glu)
|
Total number of positively charged residues (Arg + Lys)
|
Extinction Coefficients
|
Instability Index
|
Aliphatic Index
|
Grand average of hydropathicity (GRAVY)
|
Cyprinodon variegates(αA)
|
175
|
19715.82
|
5.84
|
25
|
21
|
17420
|
41.15(unstable)
|
70.74
|
-0.567
|
Cyprinodon variegates(αB)
|
164
|
19071.72
|
5.63
|
22
|
19
|
33460
|
46.19(unstable)
|
71.34
|
-0.395
|
Thunnus maccoyii(αA)
|
176
|
19978.12
|
5.70
|
25
|
21
|
15930
|
49.27(unstable)
|
65.90
|
-0.569
|
Thunnus maccoyii(αB)
|
143
|
16588.72
|
5.50
|
21
|
16
|
37470
|
47.92(unstable)
|
66.08
|
-0.548
|
Stegastus partitus(αA)
|
176
|
19890.08
|
5.84
|
25
|
21
|
17420
|
40.37(unstable)
|
69.26
|
-0.585
|
Stegatus partitus(αB)
|
162
|
19208.67
|
5.85
|
23
|
19
|
47440
|
45.56(unstable)
|
59.51
|
-0.501
|
Paralichthys olivaceus(αA)
|
176
|
20071.47
|
5.71
|
25
|
21
|
18910
|
47.25(unstable)
|
65.28
|
-0.634
|
Paralichthys olivaceus(αB)
|
164
|
19264.39
|
5.77
|
23
|
18
|
46075
|
48.78(unstable)
|
58.84
|
-0.691
|
Mastacembelus armatus(αA)
|
176
|
19959.09
|
5.76
|
25
|
20
|
17545
|
39.89(stable)
|
64.77
|
-0.559
|
Mastacembelus armatus(αB)
|
164
|
19225.76
|
6.22
|
23
|
20
|
42970
|
47.44(unstable)
|
72.50
|
-0.601
|
The Kyle-Doolittle Plots of α-crystallin of the 10 habitat-specific fish revealed no considerable differences i.e. the number of hydrophilic amino acids was found to be a little greater than the number of hydrophilic amino acids for both α- crystallin subunits as reflected from the respective positions 85-90, 105-110, 205-220,400-480, 550-599, 601-699, 750-800, 905-915 for αA- subunit of the freshwater and saltwater fish species and 100-105, 250-290, 701-720, 801-810 for αB- subunits of the fish species displaying hydrophobic sites (Fig 7-8). They bear resemblances to the hydropathy plot of bovine α- crystallin which too exhibited a similar trend, with positions 35-40, 45-59, 65-79, 123-141 in case of bovine αA- and positions 38-42, 86-98, 122-142 in bovine αB- subunit showing the presence of hydrophobic amino acids.
An analysis by TMHMM server revealed no transmembrane segments in the respective proteins of either species, indicating that α- crystallin is a cytosolic protein (Fig 9).
III. Multiple Sequence Alignment of α- Crystallin Proteins
The results of multiple sequence alignment using Clustal 2.1 and Clustal Omega programme revealed considerable amount of conservation at certain sites such as methionine residues at position 60, methionine, aspartic acid, glutamine, isoleucine, histidine,alanine, arginine, phenylalanine, glycine residues at position 16, 68, 177 for α-crystallin of freshwater fish species [Fig 10 (A), (B)]; aspartic acid, asparagine, glycine, histidine, serine, glutamic acid, methionine, valine, glutamine, arginine, lysine residues at position 114 for crystallin of saltwater fish species [Fig 10 (C) (D)]. An analysis of MSA results of crystallins of both freshwater and saltwater fish species revealed conservation at respective sites i.e. arginine, leucine, aspartic acid, asparagine,tyrosine, serine, isoleucine, lysine, alanine, proline at positions 50,97,151 respectively. The considerable amount of conservation might probably be due to the high sequence similarity among the α-crystallin of ten habitat-specific fish.
The MSA analysis of the crystallins of the fish species, along with Bos taurus revealed the presence of conserved serine residues at positions 173 for bovine as well as the fish species; at the same time presence of conserved residues proline and lysine at position 175 of bovine protein was detected.
IV. Phylogenetic Analysis
The cladograms constructed with the help of tree viewer tool (Clustal Omega). From the phylogram presented in Fig 11, it is evident that Bos taurus and the other fish species are more or less located on a single branch, except for αB crystallin of catfish, a freshwater species and αB crystallin of Sheepshead Minnow, a saltwater species which are located on a single branch, whereas α-crystallins of freshwater fish species Striped Bass, Damselfish and Zigzag eel, both saltwater fish species are located on a single branch as well.
The above results emphasises the close phylogenetic relationship among these organisms with respect to the amino acid sequence of α-crystallin protein.
V. Homology Modelling
Models of α-crystallin were generated using the homology modelling approach, by SWISS-PDB Viewer tool. The best selected templates were done based on a greater sequence identity and maximum coverage; for instance freshwater species zebrafish α- crystallin (2ygd.1.X) was selected with an identity of 53.89 and 100% coverage which bear similarities with bovine (Fig12.i.).
Similarly, on the other hand the template for catfish α- crystallin (3N3E) was selected with a sequence identity of 77.67 and 60% coverage (Fig 12.ii). On the other hand saltwater species, Zigzag eel (ID: 2YGD) α-crystallin was selected with a sequence identity of 71.10 and 99.99% coverage (Fig 12.iii.) The structures for the twenty amino acid fish sequences were overall similar, with greater sequence identities and maximum coverages; Bos taurus α-crystallin (PDB ID:2YGD)was considered as a standard template bearing closest resemblance with the crystallin protein of the ten habitat-specific fish under study (Fig 12.iv.). Such a homology might probably be due to the higher sequence similarity and sequence coverage with twenty protein sequences of fish.