Fredric Sanger determined the first protein sequence in 1953, which was insulin. There are two main methods for protein sequencing - N-terminal sequencing including the Sanger method and Edman degradation, and C-terminal sequencing using carboxypeptidases. Protein sequences can be predicted from DNA sequences after determining the gene sequence. Comparing protein sequences between organisms can provide information about evolutionary relationships and how organisms have diverged over time as mutations cause amino acid changes. Work has been done in Pakistan on sequencing various proteins.

1. Protein sequencing and evolution
Presented by:
Hafsa Jamil
Roll no: 35 1

2. Table of content:
Introduction:
• Proteins
• Protein sequencing
First protein sequence
Methods of protein sequencing
N terminal sequencing:
• Sanger method
• Edman degradation method
C terminal sequencing
Prediction from DNA sequence
Protein sequencing and evolution
Conclusion
References
2

3. What are proteins?
Class of nitrogenous organic compounds which have large
molecules composed of one or more long chains of amino
acids and are an essential part of all living organisms
3

4. Protein
sequencing:
Protein sequencing is the
practical process of determining
the amino acid sequence of all
or part of a protein or peptide.
4

5. First sequence:
First protein
sequence was
achieved by Fredric
Sanger in 1953.
He determined the
amino acid sequence
of insulin.
5

6. Sequencing
methods:
N- Terminal
sequencing
C- terminal
sequencing
Prediction
from DNA
sequence
6

7. Sanger developed the reagent 1-fluoro-2,4-
dinitrobenzene (FDNB) to label and identify the amino-
terminal amino acid residue.
Other reagents used to label the amino-terminal
residue, dansyl chloride and dabsyl chloride
After the amino-terminal residue is labeled with one of
these reagents, the polypeptide is hydrolyzed to its
constituent amino acids and the labeled amino acid is
identified.
Sanger sequencing:
7

8. 8

9. Uses Edman’s reagent phenylisothyocyanate
(PTC)
PTC combines with free N terminal amino acid
N terminal amino acid is exiced and seperated as
PTH derivative. (phenylthiodantoin derivative).
Edman degradation is carried out in a machine
called sequenter.
Edman degradation:
9

10. 10

11. Breaking
disulphide
bonds (if any)
Cleaving the
polypeptide
chain
Sequencing
the peptides
Ordering
peptide
fragments
Sequencing of larger proteins:
11

12. Less no. of
methods
available than N
terminal
sequencing
Mot common
method is the
addition of
caboxypeptidases
to a solution of
protein
Take samples at
regular interval
Determine
terminal amino
acid by analyzing
a plot of amino
acid
concentration
against time.
C-terminal sequencing
12

13. Prediction from DNA sequence:
With the development of rapid DNA sequencing methods
researchers can deduce the sequence of a polypeptide by determining
the sequence of nucleotides in the gene that codes for it
When the gene is available, sequencing the DNA can be faster and more
accurate than sequencing the protein. Most proteins are now
sequenced in this in direct way.
13

14. Protein Sequences and Evolution
Protein sequences can elucidate history of life on earth
If two organisms are closely related, the sequences of their genes and proteins
should be similar. The sequences increasingly diverge as the evolutionary
distance between two organisms increases.
Change in amino acids may be due to mutations
As more sequence information is made available in databases, evolutionary trees are
constructed based on a variety of different proteins
Analyses using the sequences of many different proteins can provide a detailed
and accurate picture of evolutionary relationships.
14

15. Work done in Pakistan:
 Structural analysis and epitope prediction of HCV E1 protein isolated in Pakistan
Sobia Idrees and Usman A Ashfaq (Idrees and Ashfaq Virology Journal2013)
Department of Bioinformatics and Biotechnology, Government College University
(GCU), Faisalabad, Pakistan
 A novel insertion mutation in the cartilage-derived morphogenetic protein-1
(CDMP1) gene underlies Grebe-type chondrodysplasia in a consanguineous
Pakistani family
Basit et al, 2008
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam
University Islamabad, Pakistan
 Mutations in WDR62 gene in Pakistani families with autosomal recessive
primary microcephaly
Kousar et al, 2011
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University,
Islamabad, Pakistan
 Molecular characterization, cloning and sequencing of coat protein gene of a
Pakistani potato leaf roll virus isolate and its phylogenetic analysis
Belal Hossain, Idrees Ahmad Nasir, Bushra Tabassum and Tayyab Husnain, 2013
Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore
15

16. Protein sequences are a rich source
of information about protein
structure and function, as well as the
evolution of life on this planet.
Sophisticated methods are being
developed to trace evolution by
analyzing the resultant slow changes
in the amino acid sequences of
homologous proteins. Protein
sequences can also be used to find
out the location of gene coding for
that protein
Conclusion:
16

17. References
• Lehninger Principles of Biochemistry (fourth edition)
David L. Nelson , Michael M. Cox
• Harper’s Illustrated Biochemistry
Robert K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell
• Methods in molecular biology volume 211
• Protein sequencing protocols
Baryan John Smith
• Fundamentals of molecular evolution
Dan Graur, Wen-Hsiung Li
• Molecules as Documents of Evolutionary History
Emile Zuckerkandl and Linus Pauling
• J Bailey - US Patent 5,807,748, 1998 - Google
• S Liang - Chinese Bulletin of Life Sciences, 1999 - europepmc.org
• https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-10-64
• https://patents.google.com/patent/US5807748A/en
17

18. 18