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Biochemistry 2/e - Garrett & Grisham
CHAPTER 5
Proteins: Their Biological
Functions and Primary Structure
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Outline
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5.1 Proteins - Linear Polymers of Amino Acids
5.2 Architecture
5.3 Many Biological Functions
5.4 May be Conjugated with Other Groups
5.7 Primary Structure Determination
5.8 Consider the Nature of Sequences
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Biochemistry 2/e - Garrett & Grisham
5.1 Proteins are Linear Polymers of Amino Acids
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Biochemistry 2/e - Garrett & Grisham
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
• Due to the double bond character, the six
atoms of the peptide bond group are
always planar!
• N partially positive; O partially negative
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane!
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
“Peptides”
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Short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
many - polypeptide
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Biochemistry 2/e - Garrett & Grisham
“Protein”
One or more polypeptide chains
• One polypeptide chain - a monomeric protein
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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
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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 - alpha - MW 2.8 million!
• beta connectin - MW of 2.1 million, with a
length of 1000 nm -it can stretch to 3000
nm!
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
The sequence of ribonuclease A
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
What forces determine the
structure?
• Primary structure - determined by
covalent bonds
• Secondary, Tertiary, Quaternary structures all determined by weak forces
• Weak forces - H-bonds, ionic interactions,
van der Waals interactions, hydrophobic
interactions
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
How to view a protein?
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backbone only
backbone plus side chains
ribbon structure
space-filling structure
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Configuration and
conformation are
not the same
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Biochemistry 2/e - Garrett & Grisham
5.3 Biological Functions of
Proteins
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Proteins are the agents of biological function
Enzymes - Ribonuclease
Regulatory proteins - Insulin
Transport proteins - Hemoglobin
Structural proteins - Collagen
Contractile proteins - Actin, Myosin
Exotic proteins - Antifreeze proteins in fish
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Biochemistry 2/e - Garrett & Grisham
The tetrameric structure of hemoglobin
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Biochemistry 2/e - Garrett & Grisham
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.
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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.
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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.
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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)
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Step 3:
Determine Amino Acid Composition
• described on pages 112,113 of G&G
• results often yield ideas for
fragmentation of the polypeptide chains
(Step 5, 6)
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Biochemistry 2/e - Garrett & Grisham
Step 4:
Identify N- and C-terminal residues
• N-terminal analysis:
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Edman's reagent
phenylisothiocyanate
derivatives are phenylthiohydantions
or PTH derivatives
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
Steps 5 and 6:
Fragmentation of the chains
• Enzymatic fragmentation
– trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation
– cyanogen bromide
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Biochemistry 2/e - Garrett & Grisham
Enzymatic Fragmentation
• Trypsin - cleavage on the C-side of Lys, Arg
• Chymotrypsin - C-side of Phe, Tyr, Trp
• Clostripain - like trypsin, but attacks Arg
more than Lys
• Staphylococcal protease
– C-side of Glu, Asp in phosphate buffer
– specific for Glu in acetate or bicarbonate buffer
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Chemical Fragmentation
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
Sequence analysis of catrocollastatin-C, a 23.6 kD
protein from the venom of Crotalus atrox
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Laboratory Synthesis of
Peptides
• Strategies are complex because of the
need to control side chain reactions
• Blocking groups must be added and
later removed
• du Vigneaud’s synthesis of oxytocin in
1953 was a milestone
• Bruce Merrifield’s solid phase method
was even more significant
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Biochemistry 2/e - Garrett & Grisham
Solid Phase Synthesis
• Carboxy terminus of a nascent peptide
is covalently anchored to an insoluble
resin
• After each addition of a residue, the
resin particles are collected by filtration
• Automation and computer control now
permit synthesis of peptides of 30
residues or more
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company