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Reginald H. Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett
Chapter 5
Proteins: Their Primary Structure and
Biological Function
Reginald Garrett & Charles Grisham • University of Virginia
Essential Question
• What structural forms do polypeptide chains
assume, how can the sequence of amino acids in
a protein be determined, and what are the
biological roles played by proteins?
Outline
• What is the fundamental structural pattern in proteins?
• What architectural arrangements characterize protein
structure?
• How are proteins isolated and purified from cells?
• How is the amino acid analysis of proteins performed?
• How is the primary structure of a protein determined?
• Can polypeptides be synthesized in the laboratory?
• What is the nature of amino acid sequences?
• Do proteins have chemical groups other than amino
acids?
• What are the many biological functions of proteins?
5.1 What Architectural Arrangements
Characterize Protein Structure?
• Proteins are classed according to shape and
and solubility
• Shape - globular or fibrous
• The four levels of protein structure are:
- Primary (1°) - sequence
- Secondary (2°) - local structures - H-bonds
- Tertiary (3°) - overall 3-dimensional shape
- Quaternary (4°) - subunit organization
5.1 What Architectural Arrangements
Characterize Protein Structure?
(a) Proteins having structural roles in cells are typically fibrous and
often water insoluble. (b) Myoglobin is a globular protein. (c)
Membrane proteins fold so that hydrophobic amino acid side
chains are exposed in their membrane-associated regions.
5.1 What Architectural Arrangements
Characterize Protein Structure?
Bovine pancreatic
ribbonuclease A
contains 124 amino
acid residues, none of
which are Trp. Four
disulfide bridges are
indicated in gold.
5.1 What Architectural Arrangements
Characterize Protein Structure?
Secondary
structures in
proteins
The α-helix and the βpleated strand are the two
principal secondary
structures found in proteins.
How to view a protein?
• The tertiary structure of a protein may be
viewed in several ways:
• Backbone only
• Backbone plus side chains
• Ribbon structure
• Space-filling structure
• Each of these is an abstraction
How to view a protein?
Folding of the polypeptide into a
compact, roughly spherical
conformation creates the tertiary
(3°) level of protein structure.
The Quaternary Level of Protein Structure
Hemoglobin is a
tetramer consisting
of two α and two β
polypeptide chains.
A Protein’s Conformation Can Be Described as Its
Overall Three-Dimensional Structure
• Be careful to distinguish the terms “conformation”
and “configuration”
• A configuration change require the breaking of a
bond.
• A protein, or any molecule, can change its
conformation by changing shape without breaking a
bond.
Figure 5.6 Configuration and
conformation are not synonymous
Imagine the conformational
possibilities for a protein in
which two of every three
bonds along its backbone
are freely rotating single
bonds.
5.2 How Are Proteins Isolated and
Purified from Cells?
• The thousands of proteins in cells can be
separated and purified on the basis of size
and electrical charge
• Proteins tend to be least soluble at their
isoelectric point
• Increasing ionic strength at first increases
the solubility of proteins (salting-in), then
decreases it (salting-out)
5.2 How Are Proteins Isolated
and Purified from Cells?
• Purification was difficult for a endogenous
protein
• First proteins studies were very abundant
• Modern cloning techniques all for production
of large quantities of specific proteins
• This process still requires that the protein
be isolated from a cell, and purified from
the other cellular components
Conditions affect protein Stability
• pH
• The wrong pH causes denaturation
• Temperature
• The wrong temperature can cause denaturation
• Presence of other proteins
• Proteases can destroy proteins
• Adsorption to surfaces
• Some proteins can be denatured upon exposure to air
• Long term storage
• Most proteins should be stored at -20°C or lower to
minimize degradation and denaturation
Protein Concentration
ELISA
Enzyme linked immunosorbent
assay
Used to determine (quantify) the
amount of protein present
Protein Concentration
Spectroscopic method for
determining protein
concentration
Beer-Lambert law
A=εcl
A280 – absorbance of F, Y, W
Protein Concentration
Colorimetric method for
determining protein
concentration
Bradford assay
Protein Purification
Salting Out
Ion Exchange Chromatography
Animation
Gel Filtration Chromatography
Animation
Affinity Chromatography
Immunoaffinity
Metal chelate
5.2 How Are Proteins Isolated and Purified
from Cells?
A typical protein purification scheme uses a series of
separation methods. Note the dramatic increase in specific
activity* of the enzyme through a series of five different
purification procedures.
*The term “specific activity” refers to the activity of the
enzyme per mg of protein.
Dialysis
Techniques, Figure 1.
A dialysis experiment.
The solution of
macromolecules is
placed in a
semipermeable
membrane bag, and the
bag is immersed in a
bathing solution.
Diffusible solutes in the
dialysis bag equilibrate
across the membrane.
SDS-PAGE
Sodium-dodecyl sulfate – Poly acrylamide gel electrophoresis
SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
Techniques, Figure 6.
A plot of protein mobility versus log of molecular
weight of individual peptides.
Two-Dimensional Gel Electrophoresis
Techniques, Figure 7.
A two-dimensional
electrophoresis separation.
Macromolecules are first
separated according to charge
by isoelectric focusing in a tube
gel. The gel containing
separated molecules is then
place on top of an SDS-PAGE
slab, and the molecules are
electrophoresed into the SDSPAGE gel, where they are
separated according to size.
Capillary Electrophoresis
2D Gel electrophoresis
5.3 How Is the Amino Acid Analysis of
Proteins Performed?
• Acid hydrolysis liberates the amino acids
of a protein
• Note that some amino acids are partially or
completely destroyed by acid hydrolysis
• Chromatographic methods are used to
separate the amino acids
• The amino acid compositions of different
proteins are different
5.4 How is the Primary Structure of a
Protein Determined?
• The sequence of amino acids in a protein is
distinctive
• Both chemical and enzymatic methodologies are
used in protein sequencing
Frederick Sanger was the first to determine
the sequence of a protein
• In 1953, Sanger 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
The sequence of insulin
The hormone insulin consists of two polypeptide chains,
A and B, held together by two disulfide (S-S) crossbridges. The A chain has 21 amino acid residues and an
intrachain disulfide; the B polypeptide contains 30 amino
acids.
Determining the Sequence – A Six-Step
Strategy
1. If more than one polypeptide chain, the chains are
separated and purified.
2. Intrachain S-S (disulfide) cross-bridges are cleaved.
3. The N-terminal and C-terminal residues are identified.
4. Each polypeptide chain is cleaved into smaller
fragments, and the composition and sequence of each
fragment is determined.
5. Step 4 is repeated, using a different cleavage procedure
to generate a different and overlapping set of peptide
fragments.
6. The overall amino acid sequence of the protein is
reconstructed from the sequences in overlapping
fragments.
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
Disulfide cleavage
Step 3:
Identify N- and C-terminal residues
• N-terminal analysis:
• Edman's reagent
• phenylisothiocyanate
• derivatives are phenylthiohydantoins (PTH
derivatives)
Dansyl Chloride
Step 3:
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 4 and 5:
Fragmentation of the chains
• Enzymatic fragmentation
• trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation
• cyanogen bromide
Polypeptide Cleavage Procedures
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
Enzymatic Fragmentation
The products of the reaction with trypsin are a mixture of
peptide fragments with C-terminal Arg or Lys residues and a
single peptide derived from the C-terminal end of the
polypeptide.
Chemical Fragmentation with Cyanogen Br
Step 6:
• 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
Edman Degradation
Reconstructing a Sequence
Compare cleavage by trypsin and staphylococcal
protease on an unknown peptide:
• Trypsin cleavage of the unknown peptide gave:
A-E-F-S-G-I-T-P-K
L-V-G-K
• Staphylococcal protease cleavage gave:
F-S-G-I-T-P-K
L-V-G-K-A-E
Reconstructing the Sequence of an
Unknown Peptide
Overlap of the two sets 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
Amino Acid Sequence Can Be
Determined by Mass Spectrometry
• Mass spectrometry separates particles on the basis
of mass-to-charge ratio
• Fragments of proteins can be generated in various
ways
• MS can also separate these fragments
Amino Acid Sequence Can Be
Determined by Mass Spectrometry
Amino Acid Sequence Can Be
Determined by Mass Spectrometry
Amino Acid Sequence Can Be
Determined by Mass Spectrometry
5.5 What is the Nature of Amino Acid
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
• Figure 5.16 illustrates the relative
frequencies of amino acids in proteins.
5.5 What is the Nature of Amino Acid
Sequences?
Frequencies of
amino acids in the
proteins of the
SWISS-PROT
database.
Computer Programs Can Align Sequences
and Discover Homology Between Proteins
Alignment of the amino acid sequences of two protein
homologs using gaps. Shown are parts of the amino acid
sequences of the catalytic subunits from the major ATPsynthesizing enzyme (ATP synthase) in a representative
archaea and a bacterium. These protein segments
encompass the nucleotide-binding site of these enzymes.
Identical residues in the two sequences are shown in red.
Introduction of a three-residue-long gap in the archaeal
sequence optimizes the alignment of the two sequences.
Blocks Substitution Matrix (BLOSUM)
• Methods for alignment and comparison of protein
sequences depend upon some quantitative measure
of how similar two sequences are.
• One way to measure similarity is to use a matrix that
assigns scores for all possible substitutions of one
amino acid for another.
• BLOSUM62 is the substitution matrix most often
used with BLAST.
• BLOSUM62 assigns a probability score for each
position in an alignment based on the frequency with
which that substitution occurs in the consensus
sequences of related proteins.
Blocks Substitution Matrix (BLOSUM)
The BLOSUM62 substitution
matrix provides scores for all
possible exchanges of one
amino acid with another.
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
Orthology in cytochrome c
The sequence of cytochrome c from more than 40
different species reveals that 28 residues are invariant.
When the sequences of a given protein from multiple
organisms are homologous, they are said to be
“orthologous”.
Related Proteins Show a Common
Evolutionary Origin
This
phylogenetic
tree depicts
the
evolutionary
relationships
among
organisms as
determined by
the similarity
of their
cytochrome c
sequences.
Related Proteins Show a Common
Evolutionary Origin
The sequence of cytochrome c is compared with an inferred
ancestral sequence represented by the base of the tree on
the previous slide. Uncertainties are denoted by question
marks.
Related Proteins Show a Common
Evolutionary Origin
The amino acid sequences of the globin chains of human
hemoglobin and myoglobin show a strong degree of
homology.
Related Proteins Show a Common
Evolutionary Origin
Compare this structure with the structures of the β-chain of
horse methemoglobin and that of sperm whale myoglobin.
Related Proteins Show a Common
Evolutionary Origin
Figure 5.21 Compare
this structure with the
structures of the αchain of horse
methemoglobin and
that of sperm whale
myoglobin.
Related Proteins Show a Common
Evolutionary Origin
This evolutionary tree
is inferred from the
homology between the
amino acid sequences
of the α–globin, βglobin, and myoglobin
chains.
Apparently Different Proteins May Share a
Common Ancestry
• Evolutionary relatedness can be inferred from
sequence homology
• Consider lysozyme and human milk α-lactalbumin
• These proteins are identical at 48 positions (out of
129 in lysozyme and 123 in human milk α-lactalbumin
• Functions of these two are not related
Apparently Different Proteins May Share a
Common Answer
The tertiary
structures of hen
egg white
lysozyme and
human αlactalbumin are
very similar.
Similar structures, but different sequence
and function
The tertiary structure of hexokinase. Compare this structure with that of G-actin
These two proteins have different sequences and different functions, but similar
tertiary structures.
Related Proteins Share a Common
Evolutionary Origin
5.7 Do Proteins Have Chemical Groups
Other Than Amino Acids?
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 Do Proteins Have Chemical Groups
Other Than Amino Acids?
5.8 What Are the Many Biological
Functions of Proteins?
• Many proteins are enzymes
• Regulatory proteins control metabolism and gene
expression
• Many DNA-binding proteins are gene-regulatory
proteins
• Transport proteins carry substances from one place
to another
• Storage proteins serve as reservoirs of amino acids
or other nutrients
5.8 What Are the Many Biological
Functions of Proteins?
• Movement is accomplished by contractile and motile
proteins
• Many proteins serve a structural role
• Proteins of signaling pathways include scaffold
proteins (adapter proteins)
• Other proteins have protective and exploitive
functions
• A few proteins have exotic functions
5.8 What Are the Many Biological
Functions of Proteins?
Questions
• You should practice questions 1-8.