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CHAPTER
5
Proteins:
Their Biological
Functions and Primary
Structure
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Proteins
are linear
polymers
aminoAcids
acids:
5.1 Proteins
are Linear
Polymers of
of Amino
The Peptide Bond
• Bond occurs between the α-amino
group of one amino acid and the
α-carboxyl group of another amino
acid
• A condensation reaction where
the elements of H20 are removed
H O
NH2 - C - C - OH
H
H
H N - C - COOH
H H
H O
H
NH2 - C - C - OH H - N - C - COOH
H
H H
The Peptide Bond!!
H O
H
NH2 - C - C N - C - COOH
H
H H
THE PEPTIDE BOND:
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
The Peptide Bond is a Resonance Structure:
H O
H
NH2 - C - C N - C - COOH
H
H O-
H H
H
NH2 - C - C N+ - C - COOH
H
H H
The coplanar nature of the Peptide Bond
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane!
“Peptides”
“Peptides”
•
•
•
•
•
•
Short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
many - polypeptide
“Protein”
“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
The tetrameric structure of hemoglobin
Proteins
Large
and
Small
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!
The
SequenceofofAmino
Amino Acids
Acids in
The
Sequence
inaa Protein
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
• Protein chains have a direction.
• By convention the N-terminus is taken to
be the beginning of a polypeptide chain.
H O
H O
H
NH2 - C - C - N - C - C -N - C - COOH
H H
H
H CH3
Glycine-Glycine-Alanine
The sequence of ribonuclease A
Architecture
of Proteins
5.2
Architecture
of Proteins
• Shape - globular, fibrous, membrane
• The levels of protein structure
- Primary - linear amino acid sequence
- Secondary - peptide backbone - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization
What
forces
determine
the
What forces determine the structure?
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
The tetrameric structure of hemoglobin
to view
a protein?
HowHow
to View
a Protein?
•
•
•
•
backbone only
backbone plus side chains
ribbon structure
space-filling structure
Configuration and
conformation are
not the same
Biological
Functions
of
Proteins:
5.3 Biological Functions of
•
•
•
•
•
•
•
Proteins
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
Storage - seed storage proteins in plants
Other Chemical Groups in Proteins:
5.4
Other
Chemical
Groups
in
Proteins may be "conjugated" with other
chemical
groups
Proteins
• If the non-amino acid part of the protein is
important to its function, it is called a
prosthetic group.
• Large organic molecules (vitamins)
comjugated to proteins are coenzymes.
• Be familiar with the terms: glycoprotein,
lipoprotein, nucleoprotein, phosphoprotein,
metalloprotein, hemoprotein, flavoprotein.
Determination
5.7Sequence
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
Determining
theSequence:
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:
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:
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 1:1:
Step
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 2:2:
Step
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 3:3:
Step
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)
Step 4:4:
Step
Identify N- and C-terminal residues
• N-terminal analysis:
–
–
–
–
Edman's reagent
phenylisothiocyanate
derivatives are phenylthiohydantions
or PTH derivatives
Step 4:4:
Step
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
Steps 5 5
& and
6: 6:
Fragmentation of the chains
• Enzymatic fragmentation
– trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation
– cyanogen bromide
Enzymatic Fragmentation
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
Chemical Fragmentation
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 7:7:
Step
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
Reconstructing
the Sequence
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
Reconstructing
the Sequence
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
NatureofofProtein
ProteinSequences
Seqences
Nature
• 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
Cc
PhylogenyofofCytochrome
Cytochrome
• 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
Laboratory Synthesis
Synthesis ofofPeptides
Laboratory
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
Laboratory
Synthesis
of Peptides
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