Download Basic Principles of Protein Chemistry

Twice Nobel Prize Winner
FREDERICK SANGER
HARD WORK IS PAID IN FORM
OF AWARDS
Prasanna Khandavilli
Curiosity is the key for Scientific
Discovery
Frederick Sanger
The Nobel Prize in Chemistry 1958
"for his work on the structure of
proteins, especially that of insulin”
The Nobel Prize in Chemistry 1980
“for their
contributions concerning
the determination of base
sequences in nucleic acids”
Frederick Sanger
Walter Gilbert
Frederick Sanger
 Born: August
13, 1918
 Place
of Birth: Rendcombe, Gloucestershire,
England
 Residence:
U.S.A./Great Britain
 Affiliation: MRC
Laboratory of Molecular
Biology, Cambridge
Basic Principles of Protein
Chemistry
Proteins - Amino Acid residues
Physical and Biological PropertiesArrangement of the Amino Acid residues
Bergmann and Niemann
Periodic arrangement of Amino Acids
Pure protein – A random mixture of similar
individuals
Chibnall
Studies on Insulin:
 Simpler
composition
 Tryptophan and Methionine absent
 Accurate analysis
Van Slyke Procedure
content of free α-amino groups
 Short Polypeptide chains
 High
Jensen & Evans:
Phenylalanine at the end of one of the chains
Molecular weight of Insulin
 Physical
methods 36,000 to 48,000
 Gutfreund 12,000
 Harfenist & Craig 6,000
Dinitrophenyl (DNP) method
1:2:4 flourodinitrobenzene (FDNB)
*Alkaline conditions
DNP method contd.
Hydrolysis of DNP protein with Acid
DNP method contd.
Extraction with Ether
Fractionation (Partition Chromatography)
Comparison of Chromatographic rates (Silica-gel
Chromatography or Paper Chromatography)
Identification and Estimation Calorimetrically
DNP labeling of Insulin
Three yellow DNP-derivatives
ε-DNP-lysine (not extracted with Ether)
 DNP-phenylalanine
 DNP-glycine

Edman phenyl isothiocyanate
method
Standard method for studying N-terminal
residues
Disulphide bridges
 Cystine
residues
to –SH derivatives
Polymerization gave insoluble products
 Reduction
How to break these Disulfide bridges?
Oxidation with Performic Acid
Precipitation of Oxidized Insulin
 Fraction A :
N-terminal residue Glycine
Acidic
Simpler composition (Lys, Arg, His, Phe,
Thr, Pro were absent)
 Fraction
B:
N-terminal residue Phenylalanine
Basic Amino acids
Acid hydrolysis of DNPPhenylalanine
Conclusions
 Position
 Only
of residues
two types of chains
 Molecular
weight 12,000
Fractionation
Paper Chromatography for Fractionation of
small peptides
Consden, Gordon, Martin & Synge worked on
pentapeptide Gramicidin-S
Fraction B studies
 Ionophoresis, Ion-exchange Chromatography,
Adsorption on Charcoal
 5-20
peptides
 Paper
Chromatography
 Analysis
of the constituent Amino Acids
Results
Conclusions
Five sequences present in Phenylalanine Chain
Problems
How the 5 sequences are joined ?
Hurdles in solving this mystery:
 Technical
difficulty in fractionating peptides
with non-polar residues (Tyr & Leu)
 Acid
lability of the bonds involving Serine
and Threonine
Solution is………
Enzymatic Hydrolysis:
Use of Proteolytic enzymes
More specific than acid hydrolysis
Proteolytic Enzymes
Pepsin – Peptide Bp3 fragment
Phe (CySO,H, Asp, Glu, Ser, Gly, Val, Leu, His)
Trypsin, Chymotrypsin studies
Fraction A studies
Problems in applying fraction B studies to
fraction A:
 Few
residues that occur only once
 Less
susceptible to enzymatic hydrolysis
 Water
soluble peptides- difficult to fractionate
on paper chromatography
Paper Ionophoresis
 pH
2.75
-COOH groups uncharged
-SO3H groups negative charge
-NH2 groups positive charge
 pH
3.5
-COOH groups charged
Results of Paper Ionophoresis
Sequence of Fraction A
Acid Hydrolysis
Ammonia produced from Amide groups on
Aspartic and Glutamic acid residues
 Position
of Amide groups:
Ionophoretic rates
Amide contents of peptides
Arrangement of Disulphide bridges
Assumptions and hypothesis:
Harfenist & Craig Mol Wt 6000
Two chains with three disulphide bridges:
Two bridges connecting the two chains
One intrachain bridge in fraction A
Disulphide interchange reaction
Disulphide interchange reaction
Contd.

Two types of disulphide interchange reactions
 In neutral & alkaline solution catalyzed by
–SH compounds
Enzymic Hydrolysis
 Chymotrypsin
action
-CySO3H.AspNH
-Leu.Val. CySO3H.Gly.Glu.Arg.Gly.Phe.Phe
Cystine peptide structure
The Structure of Insulin
Sequenced Insulin supports Protein
chemistry theories
 Hofmeister
& Fischer – Classical peptide
hypothesis
No evidence of periodicity
Random order
Unique & most significant order
Insulin from different species
Determination of Nucleotide
Sequences
 Smallest
DNA molecule - Bacteriophage
φX174 – 5,000 nucleotides
 tRNA -
75 nucleotides
32P-labelled
Fractionation of
oligonucleotides
G.G.Brownlee and B.G.Barrell method:
 Partial
degradation by enzymes
 Separation of smaller products
 Determination of sequence
 Applied to RNA sequences
Disadvantages
 Slow
and tedious
 Requires successive digestions and
fractionations
 Not easy to apply to larger DNA molecules
Copying Procedures
 C.Weissmann:
Bacteriophage Qβ
-Qβ Replicase – Complementary copy
-Pulse-labeling with radio actively labeled
nucleotides
 DNA Polymerase
substitutes Replicase
-Primer, Triphosphates containing 32P in α
position - Sanger
Copying Procedure
Primer Source
 Synthetic
Oligonucleotides
 Restriction
enzymes
Copying procedure
Results
 Short
specific regions of labeled DNA were
obtained
 Unable
to obtain individual residues for
sequencing
How to obtain individual
nucleotide residues?
Solution is ………
Incorporation of ribonucleotides in DNA
Sequence by DNA Polymerase
Splitting of ribonucleotide residues later by
action of alkali
Technique put forth by Berg, Fancher &
Chamberlin
The ‘Plus and Minus’ method
α[32P]-dNTP labeling and sequence specific
termination
J.E.Donelson - Ionophoresis of products on
acrylamide gels
The Dideoxy method
Quicker and more accurate
 φX174
 Bacteriophage G4
 Mammalian
mitochondrial DNA
Dideoxynucleoside triphosphates
 Lack
3’ hydroxyl group
 Incorporated into growing DNA chain by DNA
polymerase
 Chain terminating analogues
Dideoxy nucleotide triphosphate
Chain Termination with ddNTP
Chain-Terminating Method
Autoradiograph
DNA sequencing gel
Chain terminating method
 Problem: Requires single
stranded DNA as template
 Solution
A.J.H.Smith Exonuclease III
Fragments cloned in
plasmid vectors and Human
mitochondrial DNA
Cloning in single-stranded
Bacteriophage
 Method
 Based
to prepare template DNA
on studies of bacteriophage M 13 and
restriction fragments provided by others
Cloning
Messing – M13 Bacteriophage
Insert of β-galactosidase gene with an EcoRI
restriction enzyme site in it
 Gronenborn &
 Heidccker
96-nucleotide long restriction
fragment from M13 vector flanking EcoRI site
Cloning
Advantages
 Same
primer on all clones
 Very efficient and rapid method of
fractionating
 Each clone represents progeny of a single
molecule and is therefore pure
 No theoretical limit to the size of DNA that
could be sequenced
Bacteriophage φX174 DNA
 First
DNA sequenced by Copying procedure
 Single-stranded circular DNA
 5,386 nucleotides
 Ten genes
 Genes are overlapping
Gene Map
Reading Frames
Mammalian mitochondrial DNA
 Two
ribosomal RNAs (rRNAs)
 22-23 transfer RNAs (tRNAs)
 10-13 inner mitochondrial membrane proteins
Transcription and translation machinery of
mitochondria is different from other biological
systems
The genetic code in mitochondria
Steffans & Buse - Sequence of Subunit II of
Cytochrome Oxidase (COII) from bovine
mitochondria
Barrel, Bankier & Drouin – DNA sequence for
protein homologous to the above amino acid
sequence in human beings
Findings
 TGA - Tryptophan (not
termination codon)
 ATA – Methionine (not isoleucine)
Is it Species variation (?)
Young & Anderson-isolated bovine mtDNA
- Confirmed Uniqueness of mtDNA
mtDNA Genetic Code
Transfer RNAs
 Cytoplasmic
tRNAs:
Clover-leaf model
Invariable features
 Mammalian mt-tRNA:
Invariable features missing
Serine tRNA lacks loop of cloverleaf structure
Cytoplasmic Transfer RNAs
Wobble effect forming Family boxes
Mitochondrial Transfer RNAs
 22
tRNA genes in Mammalian mtDNA
 For
all family boxesOnly one which had a T in the position
corresponding to the third position of the
codon
 One
box
tRNA-Recognizes all codons in a family
Distribution of Protein genes
 Cytochrome oxidase
 ATPase
complex
 Cytochrome b
Gene Map of Human mtDNA
Mitochondrial DNA Conclusions
 Very
compact structure
 Reading
frames coding for proteins and rRNA
genes are flanked by tRNA genes
 Simple
model for transcription
TRENDS AND PROGRESS
IN
SEQUENCING FIELD
Trends
 1974
Conventional Sequencing Method Sanger,
Maxam & Gilbert
 1986
A regiment of scientists and technicians –
Caltech and Applied Biosystems Inc.,invented
the Automated DNA Fluorescence Sequencer.
Trends
 Craig Venter's
Sequencing Method
 In
1991, working with
Nobel laureate Hamilton
Smith, Venter's genomic
research project (TIGR)
created a new sequencing
process coined ‘shotgun
technique’.
“Trend Setter” & “Gene Hunter”
Dr. Craig Venter
Automated DNA Sequencing
 Smith
et al. 1986
 DNA molecules labeled with fluorescent dyes
 Products of dideoxy-sequencing reactions
separated by gel electrophoresis
 Dye molecules are excited by laser beam
 Fluorescent signals are amplified and detected
by Photomultiplier tubes (CCD Camera)
 Computer software identifies each nucleotide
based on the distinctive color of each dye
Automated Sequencing (Contd)
Automated Sequencing (Contd)
Genome Projects
 1999
“Celera genomics”– Rockville, Maryland
Drosophila genome
 2000
Completed Human Genome Project
http:// www.genome.gov/
 2002
Mouse Genome Project
www.informatics.jax.org/
Human Genome Project
 The
Human Genome Project
Started in 1988, Public Domain
Collaborative work between Celera Genomics
and NIH
Accomplishments:
 Identify all the approximately 35,000 genes in
human DNA
 Determine the sequences of the 3 billion chemical
bases that make up human DNA (completed July
2000)
Other Genome Databases
 A lot
of Organism specific databases at NCBI
 Allows
for Comparative Genomics studies
 Phylogenetic Analysis
 Gene Annotation
 Drug
studies
and Identification issues
therapy and Gene Therapy- Cystic
Fibrosis etc.
 DNA Vaccines
Insulin and Biotechnology
 1978: Genentech, Inc.
- Genetic engineering
techniques used to produce human insulin in E.
coli
 1983:
Genetech, Inc. licensed Eli Lily to make
insulin
Insulin Production in E.coli
3D STRUCTURE OF INSULIN
Insulin Trends
 Insulin
was first isolated from the pancreas
of cows and pigs in the early 1920s
 In
1978, a synthetic version of the human
insulin gene was constructed and inserted
into the bacterium Eschericia coli, in the
laboratory of Herbert Boyer at the
University of California at San Francisco
Insulin Trends in Medicine
 Recombinant
human insulin was developed
by Boyer's fledgling company, Genentech, in
October of 1982, the first product of modern
biotechnology
 Humulin
 Various
modes of delivering Insulin to the
Tissue
 Less Adverse
reactions, More strict glucose
control in diabetics
References
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Nobel e-Museum
The Nobel Prize Internet Archive
Britannica Nobel Prizes, Guide to the Nobel Prizes
Michigan State University, Department of Chemistry
Science Daily
http://www.geocities.com/jdelaney25/FrederickSa
nger.html
The wellcome Trust Sanger Institute
Questions and Suggestions
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