Genomic%20translations%20of%20Fenugreek%20(Trigonella%20foenum-graecum)%20to%20derive%20its%20proteomic%20insights

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Genomic translations of Fenugreek (Trigonella
foenum-graecum) to derive its proteomic insights
Geetika Jethra, Priya Gupta, Jyoti Mihra, Alka
Pawar and Sharda Choudhary
Presented By Geetika Jethra Young Profession
II National Research Centre on Seed
Spices Tabiji, Ajmer
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Seed Spices

• Indian spices include a variety of spices grown
  across the Indian subcontinent and mostly
  categorised under Apicaeae family.
• Spices are used for flavour, colour, aroma and
  preservation of food or beverages in almost all
  of the Indian Cuisines.
• But the database available on Apicaeae and
  Fabaceae family is sparsely populated with
  sequences.

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Statistics of Seed Spices
Fenugreek Cumin Coriander Fennel Celery Ajowan Anise Caraway Nigella Dill
Nucleotide 91 57 85 398 676 - 36 29 114 1749
Protein 33 38 47 4643 591 - 8 6 48 14796
Gene - - - - 18 - - - - 7
EST - - - - 1122 - - - - -
SNP - - - - - - - - - -
SSR - - - - - - - - - -
Structures - - - - - - - - - -
Genome 1 - - - 10 - - - - 3
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Fenugreek (Trigonella foenum-graecum)

• Fenugreek is commonly known as methi in Hindi
• It is an important leguminous spices and well
  known aromatic and medicinal herb.
• Fenugreek is used as both seed and leaf.
• Fenugreek seed contains carbohydrate (48),
  protein (25.5), mucilaginous matter (20), fat
  (7.9), and saponin (4.8).
• Inspite of large potential and high content of
  protein in fenugreek seeds, however, no reports
  on molecular structure predictions is available
  on Trigonella spp. native to this region.

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An Introduction to Bioinformatics

• Bioinformatics is the application of computer
  technology for the management of biological
  information.
• Computers are used to gather, store, analyse and
  integrate biological and genetic information
  which can then be applied to gene-based sequence
  determination and structure prediction.
• In the present study, Fenugreek (Trigonella
  foenum-graecum)protein models were designed and
  generated using in-silico tools.

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NCBI
National Centre for Biotechnology
Information (http//www.ncbi.nlm.nih.gov/)

• The raw data was collected from NCBI a public
  domain for the structural analysis.
• It is a comprehensive website for biologists
  including
• biology-related databases,
• tools for viewing and analyzing
• automated systems for storing and retrieval

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NCBI - Home Page (http//www.ncbi.nlm.nih.gov/)
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Calculation of Physicochemical properties

• ProtParam  A tool which allows the computation
  of various physical and chemical parameters for a
  given protein .
• (http//www.expasy.org/tools/Protparam)
• Molecular weight
• Theoretical PI
• Amino acid composition
• Atomic composition
• Instability index
• Aliphatic index and
• Grand average of hydropathicity (GRAVY)

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The Homepage of ProtParam
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Secondary Structure Prediction

• GORIV
• PSIPRED

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GOR IV
GOR is information theory based method and it
gives the information about Alpha helix, extended
strand, and beta turn in the form of propensities.
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PSIpred

• PSIPRED (bioinf.cs.ucl.ac.uk/psipred)
• ALGORITHM
• It incorporates two feed-forward neural
  networks which perform an analysis on output
  obtained from PSI-BLAST  
• (Position Specific Iterated - BLAST)

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Tertiary structure Prediction

• Swiss model
• Phyre2
• I-TASSER.

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Swiss model

• SWISS MODEL
• (http//swissmodel.expasy.org) A fully
  automated protein structure homology-modeling
  server, accessible via the ExPASy.
• It is a server used for automated comparative
  modeling of three-dimensional (3D) protein
  structures using its sequence.

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PHYRE2

• Protein Homology/AnalogY Recognition Engine
• (http//www.sbg.bio.ic.ac.uk/Phyre2/html/page.c
  gi?idindex)
• Web-based services for protein structure
  prediction.
• Generates reliable protein models when other
  widely used methods such as PSI-BLAST cannot.

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I-TASSER

• Server is an Internal service system for protein
  structure and function predictions.
• 3D models was built based on multiple-threading
  alignments performed by iterative TASSER assembly
  simulation functional insights are then derived
  by matching the predicted models with protein
  function database imbibed in it .

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Result and Discussion
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Physicochemical-properties

• ProtParam computes various physicochemical
  properties from the protein sequence. The
  parameters computed by the Server include the
  molecular weight, theoretical pI, amino acid
  composition, atomic composition, extinction
  coefficient, estimated half life, instability
  index, aliphatic index, and grand average of
  hydropathicity (GRAVY).

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Properties Fenugreek Protein
No. of Amino Acid 102
Molecular weight 11372.8
Theoretical Pi 10.38
Total no. of (-)charged residues (Asp Glu) 8
Total no. of () charged residue 15
Total number of atoms 1583
Ext. coefficient (assuming all pairs of Cys residues form cystines) 3105 M-1 cm-1
Instability index (II) 34.26
Aliphatic index 68.82
Grand average of hydropathicity (GRAVY) -0.653
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Secondary structure

• GOR IV
• PSIPRED

• Secondary structure of protein was predicted by
  the formation of alpha helix and ß-sheets.
• The results revealed that random coil (69.61)
  dominated among secondary structure elements and
  alpha helices (4.90) and extended strand
  (25.49) were also present

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Statistics of Secondary Structure
2D visualization by PSIPred
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Tertiary Structure Prediction

• Swiss model is homology based method
• Automated mode was used to get tertiary
  structure. But on the basis of structure
  validation the structure was found to be
  inappropriate.
• Next Phyre2, I-TASSER were used for 3D structure
  generation. These are threading based software.
• Verification
• Through the Structural Analysis and Verification
  Server (SAVS) the structure by Phyre2 was found
  to be better as compared to others.

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3D Structure for fenugreek protein
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Ramachandran Plot

• The Ramachandran plot is a plot of the torsional
  angles  phi and psi of each residue (amino acids)
  contained in a peptide.  By making a Ramachandran
  plot of the protein structure one can determine
  which torsion angles are lying in allowed region
  of the plot.

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Ramachandran plot by SAVES Server
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Conclusion

• The predicted protein is stable, globular and
  basic in nature having pore lining, depicting it
  as transmembrane protein
• The identified protein of fenugreek showed
  homology with the protein domain of humans and
  E-coli illustrating that the database available
  on Apicaeae and Fabaceae family are sparsely
  populated with sequences.
• FFPred predicted its functions in 2 categories
  1) Biological Process phosphate-containing
  compound metabolic process with a probability of
  95.9 and 2) Molecular function ATP binding with
  98.1 probability.

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References

• Bukhari, S.B., Bhanger, M.I. and Memon S.,
  (2008). Antioxidative Activity of Extracts from
  Fenugreek Seeds (Trigonella foenum-graecum) Pak.
  J. Anal. Environ. Chem. 9 78-83.
• Gill S.C. and Von Hippel P.H., (1989). Extinction
  coefficient. Anal. Biochem.182319- 328.
• Guermeur Y., Geourjon C., Gallinari P. And
  Deleage G. (1999). Improved performance in
  protein secondary structure prediction by
  inhomogeneous score combination. Bioinformatics,
  15 41321.
• Guruprasad K., Reddy B.V.P and Pandit M.W.,
  (1990). Correlation between stability of a
  protein and its dipeptide composition a novel
  approach for predicting in vivo stability of a
  protein from its primary sequence. Prot. Eng. 4
  155-164.

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• Harish, Gupta, A.K., Ram, K., Singh, B.,
  Phulwaria M. and Shekhawat N.S., (2011).
  Molecular and Biochemical Characterization of
  Different Accessions of Fenugreek (Trigonella
  foenum-graecum L.) Libyan Agri. Research Center
  J. International 2 150-154.
• Helambe S.S. and Dande R.P., (2012). Fenugreek
  (Trigonella foenum-graecum L.) An Overview,
  IJCPR. 2 169-187
• Ikai A.J., (1980). Thermo stability and aliphatic
  index of globular proteins. J. Biochem.88
  1895-1898.
• Jethra G., Mishra A.K., Pandey P.S. and
  Chandrasekharan H., (2012) Structure and Function
  prediction of unknown wheat protein using LOMENTS
  and I-TASSER. Indian Journal of Agricultural
  Sciences 82 86774.
• Zhang Y., (2008). Progress and challenges in
  protein structure prediction. Current Opinion in
  Structural Biology,18 342-348.

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