Research ArticleOpen Access
Structural Analysis of Cytochrome C Genes of Major Carp and Utility of Forensic Investigation
College of Fisheries, Central Agriculture University, Lembucherra, Agartala, India
|College of Fisheries|
|Central Agriculture University|
|Lembucherra, Agartala, India|
Received: May 20, 2017; Published: September 21, 2017
Citation: Rumpi G, Upadhyay AD, Roy AK, Samik A (2017) Structural Analysis of Cytochrome C Genes of Major Carp and Utility of Forensic Investigation. J Forensic crime investi 1(1): 103
The study deals with of various bioinformatics tools and in silico modelling and analysis of cytochrome c for primary, secondary and tertiary structure prediction and Ramachandran plot assessment to understand the degree of accuracy of predicted structure. Primary structure prediction and physiochemical characterization were performed of cytochrome c by computing theoretical isoelectric point (pI) 4.71, molecular weight 26029.22Da Da, instability index 43.09, aliphatic index 110.17, extinction co-efficient 41.035 Gravy 0.297. The secondary structure assessment of cytochrome protein using psipred in local carp reveals the presence greater percentage of (16) coil Regions and (8) Beta-Strand against (6) alpha helix regions. The structure information of cytochrome protein will help us to know the role of protein in Cirhinus mrigala and Labeo rohita and interaction of their ligands. The predicted structure can also be used for molecular docking studies for more insight into the structure.The resultant protein structure was evaluated by Rampage. From Ramachandran plot assessment, it was observed that the percentage of falling in most favoured region in cytochrome c is 93.7%. Based on the findings it could be concluded that further characterization of cytochrome oxidase protein is novel and will be important for evaluating how the regulation of this protein. Analysis of DNA or protein profile helps in establishing the match of the sample DNA profile under investigation with that of the standard one. If a match is found in the sample and standard one then on its bases it can be said that both belongs to the same person and absence of match indicated lack of similarity or relationship of sample with that of the standard one. Based on the above modern Computational and Bioinformatics tools and analysis we can address questions on forensic science like forensic examinations on defendant and prosecution positions during crime investigation and criminal court proceedings, Gender determination and Forensic entomology.
Keywords: Raptor X; COX; MT-CO2; Geno 3D; PSIPRED; Rampage
Cytochrome coxidase subunit 2, also known as cytochrome c oxidase polypeptide II, is a proteinthat in humans is encoded by the MT-CO2 gene. Cytochrome c oxidase subunit II, abbreviated COXII, COX2, COII, or MT-CO2, is the second subunit of oxidase. Structurally, Cytochrome is an oligomeric enzymatic complex which is a component of the respiratory chain and is involved in the transfer of electrons from cytochrome c to oxygen. In eukaryotes this enzyme complex is located in the mitochondrial inner membrane; in aerobic prokaryotes it is found in the plasma membrane. The enzyme complex consists of 3-4 subunits (prokaryotes) to up to 13 polypeptides (mammals). In Leigh's disease, there may be an abnormality or deficiency of cytochrome oxidase. Functionally, Subunit 2 (COII) transfers the electrons from cytochrome c to the catalytic subunit 1. It contains two adjacent transmembrane regions in its N-terminus and the major parts of the protein are exposed to the periplasmic or to the mitochondrial inter membrane space, respectively. COII provides the substrate-binding site and contains a copper centre called Cu (A) probably the primary acceptor in cytochrome c oxidase. An exception is the corresponding subunit of the cbb3-type oxidase which lacks the copper a redox-centre.
Several bacterial COII have a C-terminal extension that contains a covalently bound haem. The N-terminal domain of cytochrome C oxidase contains two transmembrane alpha-helices. This entry contains subunit II (CoxB) of cytochrome c oxidase. Cytochrome c oxidase is the terminal electron acceptor of mitochondria (and one of several possible acceptors in prokaryotes) in the electron transport chain of aerobic respiration. The enzyme couples the oxidation of reduced cytochrome c to the reduction of molecular oxygen to water . This process results in the pumping of four protons across the membrane that are used in the proton gradient powered synthesis of ATP. The oxidase contains two haem a cofactors and three copper atoms as well as other bound ions.The study a use of various bioinformatics tools and in silico modelling and analysis of cytochrome c for primary, secondary and tertiary structure prediction and Ramachandran plot assessment for the degree of accuracy prediction structure.
Characterization, or ''typing," of deoxyribonucleic acid (DNA) or protein for purposes of criminal investigation can be thought of as an extension of the forensic typing of blood that has been common for more than 50 years; it is actually an extension from the typing of proteins that are coded for by DNA to the typing of DNA itself. Genetically determined variation in proteins is the basis of blood groups, tissue types, and serum protein types. Developments in molecular genetics have made it possible to study the person-to-person differences in parts of DNA that are not involved in coding for proteins, and it is primarily these differences that are used in forensic applications of DNA typing to personal identification. DNA typing can be a powerful adjunct to forensic science. To understand how the protein can be used to identify individuals, it is important to understand that proteins are coded by DNA. This means that a certain level of the genetic variation that we see in different people’s DNA passes into their proteins. In fact, genetic information in the DNA is translated into amino-acid chains that make up proteins.
For the analysis of the amino acid sequence of mitochondrion cytochrome c oxidase subunit 2 or COXII of Cirrhinus mrigala are downloaded from NCBI (AFD96672), the sequence length reported to be 230 amino acids residues.
For calculation of physical and chemical parameters Expasy’s prot param server was used. For Physico-chemical characterization, theoretical isoelectric point (pI), molecular Weight, Total number of positive and negative residues and extinction Coefficient, the method given by Gill and Hippel and integrated in Expasy’s prot param server was used. Instability index, Grand average hydropathicity and aliphatic index were also computed using the Expasy’s prot param [2,3].
The secondary structure of COII was predicted by using PSIPRED (http://www.ebi.ac.uk/pdbsum/). PSI-blast based secondary structure prediction is a method used to investigate protein structure. It is a server side program, featuring a website serving as a front end interface, which can predict a protein's secondary structure (beta sheets, alpha helixes and coils) from the primary sequence. PSIPRED is available as a web service and as software. It allows modifying, but enforces freewareprovisions by forbidding for-profit distribution of the software and its results .
The aim of Geno 3d server is to make accessible to all biochemists and biologists an automated protein modelling web server to generate protein 3D model. This server is to generate protein in 3D model. The strategy used in Geno3D is comparative protein structure modelling by spatial restraints (distances and dihedral) satisfaction. Geno3D is most frequently used for homology or comparative protein structure modelling: the user provides sequence to be modelled (query) which is compared using PSI-BLAST method against a protein sequence database issue from PDB .
Homology modeling using RaptorX (http://raptorx.uchicago.edu/StructurePrediction/predict/) uses a non-linear scoring function to combine homologous information with structural information for a given template-sequence alignment. It uses to adjust relative importance of homology and structural information. Raptor uses a combination of RaptorX-Boost and Raptor-MSA to build 3D models for a target-template alignment. In the absence of good quality templates RaptorX models the alignment using an in-house free modelling programme to generate 5 models. Unaligned portions of the template are also folded by free modelling [5-7].
A Rampage use for Ramachandran plot assessment (also known as a Ramachandran diagram or a [φ,ψ] plot), originally, is a way to visualize energetically allowed regions for backbone dihedral angles ψ against φ of amino acid residues in protein structure. The geometrical and structural consistency of the predicted model was evaluated by different approaches. The φ and ψ distributions of Ramachandran plot analysis using rampage.
Expasy prot param tool was used to predict the Physico chemical properties of protein model. The ExPASy Prot param result was summarized in Table 1 for cytochrome c. The number of amino acid residues 230aa, molecular weight of the protein 26029.22Da. And the theoretical pI is 4.71 which indicate that the protein is negatively charged and that can be precipitated in acidic medium. The protein pH was calculated based on the pKa values of individual amino acid, and it can be depends on the side chains. The extinction Co-efficient, which is the quantity of the light that could be absorbed by the protein at 280 nm, is 41035. Half-life is the prediction of the time taken by the protein to reduce to half of its amount in the cell after synthesis in the cell. The server considered human, yeast and Escherichia coli cells and it was 30h>20h>and10h, respectively. As our model protein contained Met as the N- terminal residues, the half –life would be 30 h in mammalian reticulocytes (in vitro), >20h in yeast (in vivo), and > 10h in E. coli (in vivo). N –terminal arg, lys, Leu, Phe, Tyr, and Trp residues decrease the half – of the protein in E. coli .
Stability of the protein was provided by instability index. The protein showed instability index of 43.09, a protein whose instability index less than 40 was predicted as stable protein so this protein is unstable. And has half-life of >16h. The instability index is related to the half-life of the protein. The sequence contained no regions that are enriched in pro, Glu, ser and thr residues as it may result in the destabilization of the protein in eukaryotes systems. The aliphatic index is the relative Volume of the protein occupied by the aliphatic amino acids like lysine, valine, isoleucine and leucine. The aliphatic index of the protein sequence 110.17. A high aliphatic index is suggests thata protein have a wide range of temperature stability. Higher the aliphatic index greater the thermo stability of globular protein. Hydropathicity is the relative Hydrophobicity or hydrophilicity of amino acids residues present in the protein sequence . Grand average of hydropathicity (GRAVY) was found to be 0.297. The positive value indicated greater hydrophobicity and the protein is sparingly soluble in water.
|The number of amino acid residues||230aa|
|negatively charged residues(Asp+Glu)||27|
|positively charged residues(Arg+Lys)||10|
Table 1: The numbers of atoms and charged amino acid residues of cytochrome c
The secondary structure of mitochondrion Cytochrome c oxidase 2 is predicted by using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) as present in Figure 3. The secondary structure of Cytochrome oxidase revealed the presence of 16 coil Regions and 8 Beta-Strand, total 6 alpha helix regions is highlighted. On the other hand, in Labeo rohita in same protein there is found coil Regions 12, Beta-Strand 1 and total alpha helix regions 9. The secondary structure of mitochondrion Cytochrome b oxidase 2 in Labeo rohita is predicted also by PSIPRED as present in Figure 1.
The secondary structure of Cytochrome oxidase revealed the presence of 14 coil Regions and 0 Beta-Strand, total 21 alpha helix regions is highlighted. This Graph indicates that the predicted transmembrane helix or helices with interactions (Figure 2) .Helix residues are indicated as the coloured circular nodes. Interactions between residues (2:64-88 and another is 1:23-47) are indicated by edges. Helices are oriented to maximise the number of residues with predicted interactions which face one another [10,11].
(a) Cirrhinus mrigala (b) Labeo rohita
Figure 1: Secondary structure prediction of Cytochrome oxidase through PSIPRED
|Signal peptide||Not detected|
|Re-entrant helices||Not detected|
Table 2: Summary of MEMSET topology
For homology modelling template was searched by Geno 3D server and it was verified by pdb sum server (Figure 4).
The input predicted as 1 domain, The Best template 1occB selected for modelling ,p-value 9.07e-12.overall UGDT (GDT) 141(88), 160 residues are modelled,5(3%) positions predicted as disordered secondary structure: 9% H, 47% E,43% C, (whereas H indicate Helixes, E extended strand and c coil region) solvent access: 37% E, 29% M , 33% B , the solvent accessibility is divided into three states by 2 cut off values: 10% and 42% , buried for less than 10%, exposed for larger than 42% buried medium and exposed are also abbreviated as B,M and E (Figure 5,6,7,8) .
Figure 8: Modelled structure of cytochrome c oxidase subunit 2 using raptor X with side chain and backbone
From the above figure it can be seen that out of 380 aa sequences fall in most favoured region is 82.7% ,residues in allowed region 13.8%, Residues of outlier region 3.5% where as no one residues fallen in disallowed region .It also indicates that the degree of accuracy in the predicted structure. The Ramachandran plot assessment details in Figure 9, Table 3 [13,14].
|Number of residues in most favoured region||1677||(82.7%)|
|Number of residues in allowed region||280||(13.8%)|
|Number of residues of outlier region||70||(3.5%)|
|Number of residues of disallowed region||0||(0.0%)|
Table 3: Result of Ramachandran plot of cytochrome c
Primary structure analysis and physicochemical characterization was performed by calculating various indices, Gravy, molecular weight and pI. Secondary structure analysis by PSIPRED server. Structural analysis was performed after building the model using Raptor X server.3D structure information of cytochrome protein will help us to know the role of protein and interaction of their domains with their ligands. 3D structure prediction of protein requires X ray crystallography and NMR spectroscopy which is very time consuming, tedious method and generate a large amount of data creating a gap between available sequences and solved structure. In silico method of predicting 3d structure reduces this gap. Based on the findings it can be concluded that further characterization of cytochrome oxidase protein is a novel attempt and will be important for evaluating how the regulation of this protein . If in the eating product of fish something else is mixed then if we know the pure molecular weight, positive negative residue, total amino acid residues of this protein then we can determine that the fish product is not pure and we can analyse the product. A protein has only one native structure. Based on the above modern Computational and Bioinformatics tools and analysis we can address questions on forensic science like forensic examinations on defendant and prosecution positions during crime investigation and criminal court proceedings, Gender determination and Forensic entomology. In silico analysis of DNA profile may be effectively used in forensic science also .
Authors are thankful to the Dean, college of fisheries, Central Agriculture University, Lembucherra, Agartala for encouragement and support. The Financial assistant by DBT, GOI, New Delhi, India for BIF project under which this study has been carried out, is duly acknowledged.
- 1. Appaiah P, Basu P (2016) In silico designing of protein Rich in large Neutral amino acids using Bovine alpha-S1-casein for treatment of phenylketonuria. J Proteomics Bioinform 9: 287-97.
- 2. Danish I, Hariharan L, Pramodkumar GP (2014) In silico 3D structure modelling and analysis of Galactoside 2- alpha –L- fucosyltranferase 1. Research and reviews: A journal of bioinformatics 1: 01-11.
- 3. Kallberg Y (2012) Evolutionary conservation of the ribosomal biogenesis factor Rbm19/Mrd1: implications for function. PLoS One 7: e43786.
- 4. Howell N (1989) Evolutionary conservation of protein regions in the protonmotive cytochrome b and their possible roles in redox catalysis. J Mol Evol 29: 157-69.
- 5. Esposti MD, De Vries S, Crimi M, Ghelli A, Patarnello T, et al. (1993) Mitochondrial cytochrome b: evolution and structure of the protein. Biochim Biophys Acta 1143: 243-71.
- 6. Blankenship R(2009) Molecular Mechanisms of Photosynthesis. Blackwell Publishing 124-32.
- 7. Howell N (1989) Evolutionary conservation of protein regions in the proton motive cytochrome b and their possible roles in redox catalysis. J Mol Evol 29: 157-69.
- 8. Larsen Rd, Ernst LK , Nair RP (1990) Molecular cloning , sequence , and expression of human GDP –L-fucose: beta –D- galactoside 2 alpha-L-fucosyltransferase cDNA that can form the H blood group antigen. Proc Natl Acad sci USA 87: 6674-8.
- 9. Bhandari M, Kumar SRS, Kodavaty J, Raddy CVK (2013) In silico analysis and 3D structure prediction of human gastrin protein. IJARIE 2: 1-6.
- 10. Krane CM, Kishore BK (2003) Aquaporins: the membrane water channels of biological world. Biologist 50: 81-6.
- 11. Ishibashi K, Kuwahara M, Sasaki S (2003) Molecular Biology of Aquaporins. Rev Physiol Biochem Pharmaco 141: 81-6.
- 12. Källberg M, Wang H, Wang S, Peng J, Wang Z, et al. (2012) Template-based protein structure modeling using the RaptorX web server. Nature Protocols 7: 1511-22.
- 13. Müller A, MacCallum RM, Sternberg MJ (1999) Benchmarking PSI-BLAST in genome annotation. J Mol Biol 293: 1257-71.
- 14. Ghosh R, Upadhayay AD, Roy AK, Singh M (2016) In silico analysis and 3D structure prediction of mitochondrial RHO GTPase 2 protein of Danio rerio (Zebra fish) by homology modelling. J Bioinformatics and proteomics 1: 6.
- 15. Singh A, Chaube R (2014) Bioinformatic analysis, structure modelling and active site prediction from catfish Heteropneustes fossilis. IJRITCC 2: 3208-15.
- 16. Goswami AM (2015) Structural modeling and in silico analysis of non-synonymous single nucleotide polymorphisms of human 3β-hydroxysteroid dehydrogenase type 2. Meta Gene 5: 162-72.