Download Part II : Sequence Analysis What is sequence analysis?

Part II : Sequence Analysis
Paul Tan Thiam Joo
paul@bic.nus.edu.sg
Department of Biochemistry, Medicine Faculty, NUS
Institute for Infocomm Research
What is sequence analysis?
• Nucleic acids: DNA and RNA
• Proteins: amino acid composition, pI,
molecular weight, hydrophobicity.
1
Why do sequence analysis?
• Assessing potential allergenicity
(Gendel, 2002)
• Parkinson's disease (neurodegenerative)
(Tversky and Fink, 2002)
• Human Genome Project (completed in
2001)
Sequence analysis of proteins
•
•
•
•
Backtranslation
Amino acid composition
Molecular weights, pIs
Hydropathy profile
2
http://kr.expasy.org
Backtranslation
• Protein -> DNA
• Use for cloning protein of interest where it
may be present in low amount.
• Beware of codon bias and degeneracy of
codons.
3
UUU-Phe
UUC-Phe
UUA-Leu
UUG-Leu
UCU-Ser
UCC-Ser
UCA-Ser
UCG-Ser
UAU-Tyr
UAC-Tyr
UAA-Stop
UAG-Stop
UGU-Cys
UGC-Cys
UGA-Stop
UGG-Trp
CUU-Leu
CUC-Leu
CUA-Leu
CUG-Leu
CCU-Pro
CCC-Pro
CCA-Pro
CCG-Pro
CAU-His
CAC-His
CAA-Gln
CAG-Gln
CGU-Arg
CGC-Arg
CGA-Arg
CGG-Arg
AUU-Ile
AUC-Ile
AUA-Ile
AUG-Met
ACU-Thr
ACC-Thr
ACA-Thr
ACG-Thr
AAU-Asn
AAC-Asn
AAA-Lys
AAG-Lys
AGU-Ser
AGC-Ser
AGA-Arg
AGG-Arg
GUU-Val
GUC-Val
GUA-Val
GUG-Val
GCU-Ala
GCC-Ala
GCA-Ala
GCG-Ala
GAU-Asp
GAC-Asp
GAA-Glu
GAG-Glu
GGU-Gly
GGC-Gly
GGA-Gly
GGG-Gly
Biased codon usage
Amino acid
Leu
Val
Codon
UUA
UUG
CUU
CUC
CUA
CUG
GUU
GUC
GUA
GUG
Bacteria
Yeast
Fruit Fly
Human
Preferred
Preferred
Preferred
Preferred
Preferred
Preferred
Preferred
Preferred
4
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 -> absent
(Tekaia et al., 2002).
– predict structural class (Luo et al., 2002).
Nonpolar amino acids (FILMWAV)
5
Polar uncharged (S-Q+T-N+Y-)
Polar charged (KHERD)
6
Unique Properties
Protein functions from specific residues
•
•
•
•
•
C
G
H
KR
P
Disulphide-rich, zinc fingers
Collagens
Histidine-rich glycoprotein
Nuclear proteins, nuclear localisation
Collagen, filaments
7
8
Molecular weights, pIs
• Aid in designing of purification experiments
e.g. SDS-PAGE, IEF, 2-Dimensional Gel,
Column chromatography etc.
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.
9
A sliding window
M K F F L M C L I I F P I M G V L G
Signal region
Alpha-helix
10
Alpha-helix
Beta-sheet
Alpha-helix
A schematic representation of a 3-D structure of a
scorpion toxin
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.
11
Venn Diagram of the 20 amino
acid physiochemical properties
Hydropathy Profiles
• Basic ranking: internal {FILMV}, external
{DEHKNQR}, ambivalent {ACGPSTWY}
12
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)
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: 87–98.
Luo RY, Feng ZP, Liu JK. (2002) Eur J Biochem 2002 269(17):42194225
Tekaia F, Yeramian E, Dujon B. (2002) Gene 297:51-60.
Tversky VN, Fink AL. (2002) FEBS Lett 522(1-3):9-13.
EXPASY http://cn.expasy.org/
13