Part II : Sequence Analysis

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Part II Sequence Analysis
Paul Tan Thiam Joo paul_at_bic.nus.edu.sg Department
of Biochemistry, Medicine Faculty, NUS Institute
for Infocomm Research
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What is sequence analysis?

• Nucleic acids DNA and RNA
• Proteins amino acid composition, pI, molecular
  weight, hydrophobicity.

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Why do sequence analysis?

• Assessing potential allergenicity (Gendel, 2002)
• Parkinson's disease (neurodegenerative) (Tversky
  and Fink, 2002)
• Human Genome Project (completed in 2001)

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Sequence analysis of proteins

• Backtranslation
• Amino acid composition
• Molecular weights, pIs
• Hydropathy profile

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http//kr.expasy.org
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Backtranslation

• Protein -gt DNA
• Use for cloning protein of interest where it may
  be present in low amount.
• Beware of codon bias and degeneracy of codons.

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UUU-Phe UCU-Ser UAU-Tyr UGU-Cys
UUC-Phe UCC-Ser UAC-Tyr UGC-Cys
UUA-Leu UCA-Ser UAA-Stop UGA-Stop
UUG-Leu UCG-Ser UAG-Stop UGG-Trp
CUU-Leu CCU-Pro CAU-His CGU-Arg
CUC-Leu CCC-Pro CAC-His CGC-Arg
CUA-Leu CCA-Pro CAA-Gln CGA-Arg
CUG-Leu CCG-Pro CAG-Gln CGG-Arg
AUU-Ile ACU-Thr AAU-Asn AGU-Ser
AUC-Ile ACC-Thr AAC-Asn AGC-Ser
AUA-Ile ACA-Thr AAA-Lys AGA-Arg
AUG-Met ACG-Thr AAG-Lys AGG-Arg
GUU-Val GCU-Ala GAU-Asp GGU-Gly
GUC-Val GCC-Ala GAC-Asp GGC-Gly
GUA-Val GCA-Ala GAA-Glu GGA-Gly
GUG-Val GCG-Ala GAG-Glu GGG-Gly
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Biased codon usage
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Amino Acid Composition

• Determine the percentages of amino acid residues
  present in a protein molecule.
• Uses
• determine the lifestyles of organisms high
  percentages of Glutamate (- charge) and both
  Lysine and Arginine ( charge) in
  hyperthermophiles vs. mesophiles -gt absent
  (Tekaia et al., 2002).
• predict structural class (Luo et al., 2002).

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Nonpolar amino acids (FILMWAV)
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Polar uncharged (S-QT-NY-)
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Polar charged (KHERD)
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Unique Properties
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Protein functions from specific residues

• C Disulphide-rich, zinc fingers
• G Collagens
• H Histidine-rich glycoprotein
• KR Nuclear proteins, nuclear localisation
• P Collagen, filaments

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Molecular weights, pIs

• Aid in designing of purification experiments e.g.
  SDS-PAGE, IEF, 2-Dimensional Gel, Column
  chromatography etc.

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Hydropathy Profiles

• Hydropathy - describe the hydrophobicity and
  hydrophilicity of a protein sequence.
• A graph in which hydropathy values are calculated
  within a sliding window and plotted for each
  residue in a protein sequence.

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A sliding window
M K F F L M C L I I F P I M G V L G
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Signal region
Alpha-helix
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A schematic representation of a 3-D structure of
a scorpion toxin
Alpha-helix
Beta-sheet
Alpha-helix
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Hydropathy Profiles

• Hydropathy scale - each amino acid is assigned a
  value reflecting its relative hydrophobicity and
  hydrophilicity.
• 2 broad classes of scales
• Environmental characteristics of protein
  residues.
• Experimental measurements of amino acid
  physiochemical properties.

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Venn Diagram of the 20 amino acid physiochemical
properties
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Hydropathy Profiles

• Basic ranking internal FILMV, external
  DEHKNQR, ambivalent ACGPSTWY

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Hydropathy Profiles

• Detect possible transmembrane domains
  (consecutive 20-25 runs of hydrophobic amino
  acids).
• Hydrophobic protein cores
• Predict neurotoxicity in snake Phospholipases A2
  (Kini and Iwanaga, 1986)

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References

• Kini RM, Iwanaga S. (1986) Toxicon 24(6)527-541.
• Rehm BH. (2001) Appl Microbiol Biotechnol.
  57(5-6)579-92.
• Weir M, Swindells M, OveringTon J. (2001) Trends
  Biotechnol 19(10 Suppl)S61-6.
• Gendel S. M. (2002) Ann. N.Y. Acad. Sci. 964
  8798.
• Luo RY, Feng ZP, Liu JK. (2002) Eur J Biochem
  2002 269(17)4219-4225
• Tekaia F, Yeramian E, Dujon B. (2002) Gene
  29751-60.
• Tversky VN, Fink AL. (2002) FEBS Lett
  522(1-3)9-13.
• EXPASY http//cn.expasy.org/