Garrett2e Chapter05

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
All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company, 6277
Sea Harbor Drive, Orlando, Florida 32887-6777
Copyright © 1999 by Harcourt Brace & Company
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
• 5.7 Primary Structure Determination
• 5.8 Consider the Nature of Sequences

5.1 Proteins are Linear Polymers of Amino Acids

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 Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!


“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”
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 - 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 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


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

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
• Structural proteins - Collagen
• Contractile proteins - Actin, Myosin
• Exotic proteins - Antifreeze proteins in fish

The tetrameric structure of hemoglobin

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.

5.7 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


• 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.


• 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
• described on pages 112,113 of G&G
• 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


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 - 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
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

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


• 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




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
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

5.1 Proteins are Linear Polymers of Amino Acids

Copyright © 1999 by Harcourt Brace & Company

The Peptide Bond

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

The Coplanar Nature of the Peptide Bond

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

Proteins - Large and Small

Copyright © 1999 by Harcourt Brace & Company

The Sequence of Amino Acids

The sequence of ribonuclease A

Copyright © 1999 by Harcourt Brace & Company

5.2 Architecture of Proteins

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

What forces determine the

Copyright © 1999 by Harcourt Brace & Company

How to view a protein?

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Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

5.3 Biological Functions of

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Copyright © 1999 by Harcourt Brace & Company

5.4 Other Chemical Groups in

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5.7 Sequence Determination

Copyright © 1999 by Harcourt Brace & Company

Insulin consists of two

The sequence shown is

Copyright © 1999 by Harcourt Brace & Company

Determining the Sequence

• 1. If more than one polypeptide chain,

Copyright © 1999 by Harcourt Brace & Company

Determining the Sequence

• 5. Cleave each chain into smaller

Copyright © 1999 by Harcourt Brace & Company

Determining the Sequence

• 7. Reconstruct the sequence of the

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

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Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

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Copyright © 1999 by Harcourt Brace & Company

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Reconstructing the Sequence

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