CHAPTER 2 Protein
Section 2 Proteins,Their Biological
Functions and Primary Structure
Outline
5.1 Proteins - Linear Polymers of Amino Acids
5.2 Architecture
5.3 Many Biological Functions
5.4 May be Conjugated with Other Groups
5.6 Purification
5.7 Primary Structure Determination
5.8 Consider the Nature of Sequences
5.1 Proteins are Linear Polymers of Amino Acids
N partially positive;
O partially negative
The Peptide Bond
is usually found in the trans conformation
has partial (40%) double bond character
is about 0.133 nm long - shorter than a typical
single bond but longer than a double bond
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane!
Due to the double bond character,the six
atoms of the peptide bond group are always planar!
Peptides Classification
Peptides --Short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
many - polypeptide
Proteins are composed of one or
more polypeptide chains
Protein---One or more polypeptide chains
One polypeptide chain - a monomeric protein
More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
Hemoglobin,for example,is a heterotetramer
It has two alpha chains and two beta chains
Proteins - Large and Small
Insulin - A chain of 21 residues,B chain of 30
residues -total mol,wt,of 5,733
Glutamine synthetase - 12 subunits of 468
residues each - total mol,wt,of 600,000
Connectin proteins(肌联蛋白） - alpha - MW
2.8 million!
beta connectin - MW of 2.1 million,with a
length of 1000 nm -it can stretch to 3000 nm!
Acid hydrolysis of proteins
6N HCl 110℃ 24 or 48 or 72hr
Trp
Ser Thr
Asn Gln
Amino acid analysis
Table 5.2
Amino Acid Composition of Some Selected Proteins
Values expressed are percent representation of each amino
acid,Proteins*
Amino Acid RNase ADH Mb Histone H3 Collagen
Ala 6.9 7.5 9.8 13.3 11.7
Arg 3.7 3.2 1.7 13.3 4.9
Asn 7.6 2.1 2.0 0.7 1.0
Asp 4.1 4.5 5.0 3.0 3.0
The Sequence of Amino Acids in
a Protein
is a unique characteristic of every protein
is encoded by the nucleotide sequence of
DNA
is thus a form of genetic information
is read from the amino terminus to the
carboxyl terminus
The sequence of ribonuclease A
5.2 Architecture of Proteins
Shape - globular or fibrous
The levels of protein structure
- Primary - sequence
- Secondary - local structures - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization
The tetrameric structure of hemoglobin
How to view a protein?
backbone only
backbone plus side chains
ribbon structure
space-filling structure
Configuration and
conformation are
not the same
5.3 Biological Functions of
Proteins
Proteins are the agents of biological function
Enzymes - Ribonuclease
Regulatory proteins - Insulin
Transport proteins - Hemoglobin
Nutrient and Storage Proteins
Structural proteins - Collagen
Contractile proteins – Actin（ 肌动蛋白）
Myosin（ 肌球蛋白）
Scaffold Proteins 支架蛋白
(Adapter Proteins)接头蛋白
modular organization （ 组件组织）
Exotic proteins -
Monellin,Thaumatin
Protective and Exploitive Proteins -
immunoglobulins or antibodies
Antifreeze proteins in fish
ricin
5.4 Other Chemical Groups in
Proteins
Proteins may be "conjugated" with other
chemical groups
If the non-amino acid part of the protein is
important to its function,it is called a
prosthetic group.
Be familiar with the terms,glycoprotein,
lipoprotein,nucleoprotein,phosphoprotein,
metalloprotein,hemoprotein,flavoprotein.
5.5 Reactions of Peptides and Proteins
5.6 Purification of Protein Mixtures
on the basis of,
size and electrical charge
Separation Methods
size exclusion chromatography,
ultrafiltration,and ultracentrifugation
(see Appendix),
A Typical Protein Purification
Scheme
Table 5.5
Example of a Protein Purification Scheme,Purification of
the Enzyme Xanthine Dehydrogenase from a Fungus
Fraction Volume(mL) TotalProtein
(mg)
Total
Activity*
Specific
Activity?
Percent
Recovery?
1,Crude extract
2,Salt precipitate
3,Ion exchange
chromatography
4,Molecular sieve
chromatography
5,Immunoaffinity
chromatography§
3,800
165
65
4
6
22,800
2,800
100
14.5
1.8
2,460
1,190
720
555
275
0.108
0.425
7.2
38.3
152
100
48
29
23
11
5.7 Sequence Determination
Frederick Sanger was the first - in 1953,he
sequenced the two chains of insulin.
Sanger's results established that all of the
molecules of a given protein have the
same sequence.
Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in
the gene
Insulin consists of two
polypeptide chains,A and B,
held together by two disulfide
bonds,The A chain has 21
residues and the B chain has
30 residues.
The sequence shown is
that of bovine insulin.
Determining the Sequence
An Eight Step Strategy
1,If more than one polypeptide chain,separate.
2,Cleave (reduce) disulfide bridges
3,Determine composition of each chain
4,Determine N- and C-terminal residues
Determining the Sequence
An Eight Step Strategy
5,Cleave each chain into smaller
fragments and determine the
sequence of each chain
6,Repeat step 5,using a different
cleavage procedure to generate a
different set of fragments.
Determining the Sequence
An Eight Step Strategy
7,Reconstruct the sequence of the
protein from the sequences of
overlapping fragments
8,Determine the positions of the
disulfide crosslinks
Step 1:
Separation of chains
Subunit interactions depend on weak forces
Separation is achieved with:
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually
ammonium sulfate)
Step 2:
Cleavage of Disulfide bridges
Performic acid oxidation
Sulfhydryl reducing agents
- mercaptoethanol
- dithiothreitol or dithioerythritol
- to prevent recombination,follow with an
alkylating agent like iodoacetate
Step 3:
Determine Amino Acid Composition
results often yield ideas for
fragmentation of the polypeptide chains
(Step 5,6)
Step 4:
Identify N- and C-terminal residues
N-terminal analysis:
– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantions
– or PTH derivatives
PITC
DNP-peptide
(yellow)
DNP-aa
(yellow)
DNFB
Step 4:
Identify N- and C-terminal residues
C-terminal analysis
– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue except
Pro,Arg,and Lys
– Carboxypeptidase B (hog pancreas) only works
on Arg and Lys
Steps 5 and 6:
Fragmentation of the chains
Enzymatic fragmentation
– trypsin,chymotrypsin,clostripain,
staphylococcal protease
Chemical fragmentation
– cyanogen bromide
Enzymatic Fragmentation
Trypsin（ 胰蛋白酶 ） - cleavage on the C-side
of Lys,Arg
Chymotrypsin（ 胰凝乳蛋白酶 ） - C-side of
Phe,Tyr,Trp
Clostripain （ 梭菌蛋白酶） - like trypsin,but
attacks Arg more than Lys
Staphylococcal protease （ 葡萄球菌蛋白酶）
– C-side of Glu,Asp in phosphate buffer
– specific for Glu in acetate or bicarbonate buffer
Chemical Fragmentation
Cyanogen bromide
CNBr acts only on methionine residues
CNBr is useful because proteins usually
have only a few Met residues
see Fig,5.21 for mechanism
be able to recognize the results!
– a peptide with a C-terminal homoserine
lactone
Step 7:
Reconstructing the Sequence
Use two or more fragmentation agents in
separate fragmentation experiments
Sequence all the peptides produced
(usually by Edman degradation)
Compare and align overlapping peptide
sequences to learn the sequence of the
original polypeptide chain
Reconstructing the Sequence
Compare cleavage by trypsin and
staphylococcal protease on a typical
peptide:
Trypsin cleavage,
A-E-F-S-G-I-T-P-K L-V-G-K
Staphylococcal protease,
F-S-G-I-T-P-K L-V-G-K-A-E
Reconstructing the Sequence
The correct overlap of fragments,
L-V-G-K A-E-F-S-G-I-T-P-K
L-V-G-K-A-E F-S-G-I-T-P-K
Correct sequence,
L-V-G-K-A-E-F-S-G-I-T-P-K
Sequence analysis of catrocollastatin-C,a 23.6
kD protein from the venom of Crotalus atrox
Nature of Protein Sequences
Sequences and composition reflect the
function of the protein
Membrane proteins have more
hydrophobic residues,whereas fibrous
proteins may have atypical sequences
Homologous proteins from different
organisms have homologous sequences
e.g.,cytochrome c is highly conserved
Phylogeny of Cytochrome c
The number of amino acid differences between
two cytochrome c sequences is proportional to
the phylogenetic difference between the species
from which they are derived
This observation can be used to build
phylogenetic trees of proteins
This is the basis for studies of molecular
evolution
系统发生
Appendix to Chapter 5
Protein Techniques
Homogenization and solubilization（ 匀浆及溶解）
precipitation （ 沉淀 ）
Acids or alkali
Salt concentration （ salting out,salting in)
eg,Ammonium sulfate ( (NH4)2SO4 )
.
盐析（ （ NH4） 2SO4）
聚合
Organic solvents
Heavy metal ions
生物碱
Size Exclusion Chromatography
Ion-exchange chromatography
Separation depends on overall (net) charge
of the protein molecules as a mixture
interacts with polyanionic or polycationic
beads
Peptides Can Be Distinguished by
Their Ionization Behavior
Dialysis and Ultrafiltration
Electrophoresis:
Separation depends on net charge and
relative mobility of the protein molecules as
a mixture is subjected to an electric field at
constant pH.
① Visualization of proteins in gels
Coomassie brilliant blue
Silver salt
Radioactive?X-ray film
② Classification
A,Native PAGE (polyacrylamide gel
electrophoresis)
B,SDS-PAGE (sodium dodecyl sulfate PAGE)
SDS-Polyacrylamide Gel Electrophoresis
C,Isoelectric focusing:
Separation depends on net charge as the
protein molecules undergo
electrophoresis in a pH gradient until
each reaches the point at which pI=pH
D,Two-dimensional gel electrophoresis
=Isoelectic focusing + SDS-PAGE
Assay of proteins
① electrophoresis
② western blot analysis
Affinity Chromatography
Separation depends on specific,noncovalent
interactions between a protein molecule
and a ligand that is immobilized (on
column or beads).
Hydrophobic Interaction Chromatography
(HIC)
High-Performance Liquid Chromatography
Ultracentrifugation
60000~80000转 /分重力 60万～ 80万倍? Ultracentrifugation
enzymelinked immunosorbent assay (酶联免疫分析 ELISA)
Section 2 Proteins,Their Biological
Functions and Primary Structure
Outline
5.1 Proteins - Linear Polymers of Amino Acids
5.2 Architecture
5.3 Many Biological Functions
5.4 May be Conjugated with Other Groups
5.6 Purification
5.7 Primary Structure Determination
5.8 Consider the Nature of Sequences
5.1 Proteins are Linear Polymers of Amino Acids
N partially positive;
O partially negative
The Peptide Bond
is usually found in the trans conformation
has partial (40%) double bond character
is about 0.133 nm long - shorter than a typical
single bond but longer than a double bond
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane!
Due to the double bond character,the six
atoms of the peptide bond group are always planar!
Peptides Classification
Peptides --Short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
many - polypeptide
Proteins are composed of one or
more polypeptide chains
Protein---One or more polypeptide chains
One polypeptide chain - a monomeric protein
More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
Hemoglobin,for example,is a heterotetramer
It has two alpha chains and two beta chains
Proteins - Large and Small
Insulin - A chain of 21 residues,B chain of 30
residues -total mol,wt,of 5,733
Glutamine synthetase - 12 subunits of 468
residues each - total mol,wt,of 600,000
Connectin proteins(肌联蛋白） - alpha - MW
2.8 million!
beta connectin - MW of 2.1 million,with a
length of 1000 nm -it can stretch to 3000 nm!
Acid hydrolysis of proteins
6N HCl 110℃ 24 or 48 or 72hr
Trp
Ser Thr
Asn Gln
Amino acid analysis
Table 5.2
Amino Acid Composition of Some Selected Proteins
Values expressed are percent representation of each amino
acid,Proteins*
Amino Acid RNase ADH Mb Histone H3 Collagen
Ala 6.9 7.5 9.8 13.3 11.7
Arg 3.7 3.2 1.7 13.3 4.9
Asn 7.6 2.1 2.0 0.7 1.0
Asp 4.1 4.5 5.0 3.0 3.0
The Sequence of Amino Acids in
a Protein
is a unique characteristic of every protein
is encoded by the nucleotide sequence of
DNA
is thus a form of genetic information
is read from the amino terminus to the
carboxyl terminus
The sequence of ribonuclease A
5.2 Architecture of Proteins
Shape - globular or fibrous
The levels of protein structure
- Primary - sequence
- Secondary - local structures - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization
The tetrameric structure of hemoglobin
How to view a protein?
backbone only
backbone plus side chains
ribbon structure
space-filling structure
Configuration and
conformation are
not the same
5.3 Biological Functions of
Proteins
Proteins are the agents of biological function
Enzymes - Ribonuclease
Regulatory proteins - Insulin
Transport proteins - Hemoglobin
Nutrient and Storage Proteins
Structural proteins - Collagen
Contractile proteins – Actin（ 肌动蛋白）
Myosin（ 肌球蛋白）
Scaffold Proteins 支架蛋白
(Adapter Proteins)接头蛋白
modular organization （ 组件组织）
Exotic proteins -
Monellin,Thaumatin
Protective and Exploitive Proteins -
immunoglobulins or antibodies
Antifreeze proteins in fish
ricin
5.4 Other Chemical Groups in
Proteins
Proteins may be "conjugated" with other
chemical groups
If the non-amino acid part of the protein is
important to its function,it is called a
prosthetic group.
Be familiar with the terms,glycoprotein,
lipoprotein,nucleoprotein,phosphoprotein,
metalloprotein,hemoprotein,flavoprotein.
5.5 Reactions of Peptides and Proteins
5.6 Purification of Protein Mixtures
on the basis of,
size and electrical charge
Separation Methods
size exclusion chromatography,
ultrafiltration,and ultracentrifugation
(see Appendix),
A Typical Protein Purification
Scheme
Table 5.5
Example of a Protein Purification Scheme,Purification of
the Enzyme Xanthine Dehydrogenase from a Fungus
Fraction Volume(mL) TotalProtein
(mg)
Total
Activity*
Specific
Activity?
Percent
Recovery?
1,Crude extract
2,Salt precipitate
3,Ion exchange
chromatography
4,Molecular sieve
chromatography
5,Immunoaffinity
chromatography§
3,800
165
65
4
6
22,800
2,800
100
14.5
1.8
2,460
1,190
720
555
275
0.108
0.425
7.2
38.3
152
100
48
29
23
11
5.7 Sequence Determination
Frederick Sanger was the first - in 1953,he
sequenced the two chains of insulin.
Sanger's results established that all of the
molecules of a given protein have the
same sequence.
Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in
the gene
Insulin consists of two
polypeptide chains,A and B,
held together by two disulfide
bonds,The A chain has 21
residues and the B chain has
30 residues.
The sequence shown is
that of bovine insulin.
Determining the Sequence
An Eight Step Strategy
1,If more than one polypeptide chain,separate.
2,Cleave (reduce) disulfide bridges
3,Determine composition of each chain
4,Determine N- and C-terminal residues
Determining the Sequence
An Eight Step Strategy
5,Cleave each chain into smaller
fragments and determine the
sequence of each chain
6,Repeat step 5,using a different
cleavage procedure to generate a
different set of fragments.
Determining the Sequence
An Eight Step Strategy
7,Reconstruct the sequence of the
protein from the sequences of
overlapping fragments
8,Determine the positions of the
disulfide crosslinks
Step 1:
Separation of chains
Subunit interactions depend on weak forces
Separation is achieved with:
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually
ammonium sulfate)
Step 2:
Cleavage of Disulfide bridges
Performic acid oxidation
Sulfhydryl reducing agents
- mercaptoethanol
- dithiothreitol or dithioerythritol
- to prevent recombination,follow with an
alkylating agent like iodoacetate
Step 3:
Determine Amino Acid Composition
results often yield ideas for
fragmentation of the polypeptide chains
(Step 5,6)
Step 4:
Identify N- and C-terminal residues
N-terminal analysis:
– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantions
– or PTH derivatives
PITC
DNP-peptide
(yellow)
DNP-aa
(yellow)
DNFB
Step 4:
Identify N- and C-terminal residues
C-terminal analysis
– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue except
Pro,Arg,and Lys
– Carboxypeptidase B (hog pancreas) only works
on Arg and Lys
Steps 5 and 6:
Fragmentation of the chains
Enzymatic fragmentation
– trypsin,chymotrypsin,clostripain,
staphylococcal protease
Chemical fragmentation
– cyanogen bromide
Enzymatic Fragmentation
Trypsin（ 胰蛋白酶 ） - cleavage on the C-side
of Lys,Arg
Chymotrypsin（ 胰凝乳蛋白酶 ） - C-side of
Phe,Tyr,Trp
Clostripain （ 梭菌蛋白酶） - like trypsin,but
attacks Arg more than Lys
Staphylococcal protease （ 葡萄球菌蛋白酶）
– C-side of Glu,Asp in phosphate buffer
– specific for Glu in acetate or bicarbonate buffer
Chemical Fragmentation
Cyanogen bromide
CNBr acts only on methionine residues
CNBr is useful because proteins usually
have only a few Met residues
see Fig,5.21 for mechanism
be able to recognize the results!
– a peptide with a C-terminal homoserine
lactone
Step 7:
Reconstructing the Sequence
Use two or more fragmentation agents in
separate fragmentation experiments
Sequence all the peptides produced
(usually by Edman degradation)
Compare and align overlapping peptide
sequences to learn the sequence of the
original polypeptide chain
Reconstructing the Sequence
Compare cleavage by trypsin and
staphylococcal protease on a typical
peptide:
Trypsin cleavage,
A-E-F-S-G-I-T-P-K L-V-G-K
Staphylococcal protease,
F-S-G-I-T-P-K L-V-G-K-A-E
Reconstructing the Sequence
The correct overlap of fragments,
L-V-G-K A-E-F-S-G-I-T-P-K
L-V-G-K-A-E F-S-G-I-T-P-K
Correct sequence,
L-V-G-K-A-E-F-S-G-I-T-P-K
Sequence analysis of catrocollastatin-C,a 23.6
kD protein from the venom of Crotalus atrox
Nature of Protein Sequences
Sequences and composition reflect the
function of the protein
Membrane proteins have more
hydrophobic residues,whereas fibrous
proteins may have atypical sequences
Homologous proteins from different
organisms have homologous sequences
e.g.,cytochrome c is highly conserved
Phylogeny of Cytochrome c
The number of amino acid differences between
two cytochrome c sequences is proportional to
the phylogenetic difference between the species
from which they are derived
This observation can be used to build
phylogenetic trees of proteins
This is the basis for studies of molecular
evolution
系统发生
Appendix to Chapter 5
Protein Techniques
Homogenization and solubilization（ 匀浆及溶解）
precipitation （ 沉淀 ）
Acids or alkali
Salt concentration （ salting out,salting in)
eg,Ammonium sulfate ( (NH4)2SO4 )
.
盐析（ （ NH4） 2SO4）
聚合
Organic solvents
Heavy metal ions
生物碱
Size Exclusion Chromatography
Ion-exchange chromatography
Separation depends on overall (net) charge
of the protein molecules as a mixture
interacts with polyanionic or polycationic
beads
Peptides Can Be Distinguished by
Their Ionization Behavior
Dialysis and Ultrafiltration
Electrophoresis:
Separation depends on net charge and
relative mobility of the protein molecules as
a mixture is subjected to an electric field at
constant pH.
① Visualization of proteins in gels
Coomassie brilliant blue
Silver salt
Radioactive?X-ray film
② Classification
A,Native PAGE (polyacrylamide gel
electrophoresis)
B,SDS-PAGE (sodium dodecyl sulfate PAGE)
SDS-Polyacrylamide Gel Electrophoresis
C,Isoelectric focusing:
Separation depends on net charge as the
protein molecules undergo
electrophoresis in a pH gradient until
each reaches the point at which pI=pH
D,Two-dimensional gel electrophoresis
=Isoelectic focusing + SDS-PAGE
Assay of proteins
① electrophoresis
② western blot analysis
Affinity Chromatography
Separation depends on specific,noncovalent
interactions between a protein molecule
and a ligand that is immobilized (on
column or beads).
Hydrophobic Interaction Chromatography
(HIC)
High-Performance Liquid Chromatography
Ultracentrifugation
60000~80000转 /分重力 60万～ 80万倍? Ultracentrifugation
enzymelinked immunosorbent assay (酶联免疫分析 ELISA)
