CHEM 3303.
02
Biochemistry
Chapter 3:
Amino Acids, Peptides,
and Proteins
Knowledge/ skills
Entire chapter- All except protein
sequencing to small peptides can be
chemically sequenced (pg 97 to 104)
Amino acids- structure and properties
Peptides and proteins
Working with proteins
Introduction to the covalent structure of
proteins
Protein sequences and evolution
Proteins- the most abundant
macromolecule
Proteins exhibit enormous diversity and
range in size from relatively small peptides
to huge polymers
Proteins are the molecular instruments
through which genetic information is
expressed
Relatively simple monomeric units are the
building blocks of thousands of different
proteins
All proteins are constructed from the same
ubiquitous set of 20 amino acids that are
covalently linked in linear sequences
Proteins- the most abundant
macromolecule
Each amino acid has a side chain with a
distinctive chemical group with distinctive
properties
The 20 amino acids can be regarded as
the alphabet in which the language of
protein structure is written
Proteins with strikingly different
properties/ activities can be made by
joining the same 20 amino acids in
different combinations and sequences
General structure
of an amino acid-
common to all except
proline; all are amino Carbonyl group
acids
R group or side
Amino group chain is different
in each amino
carbon acid; vary in
structure, size and
electric charge
Amino acids
R- groups influence solubility in water
In addition to the 20 common, there are less common
ones
Common amino acids are assigned a 3 letter
abbreviation and a one letter symbol
The carbon is chiral in all common amino acids except
for glycine
Amino acid residues in protein molecules are almost
exclusively L stereoisomers- exceptions in some small
peptides of bacterial cell walls and some peptide
antibiotics
L stereoisomer is synthesized because the enzyme
active site is asymmetric so the reactions catalyzed are
stereospecific
Amino acids can be classified by R-
groups- 5 main classes
Amino acids can be classified by R-
groups- 5 main classes
Enantiomers
optically active
Nonpolar,
hydrophobic, tend to
cluster together by
hydrophobic
interactions
*thioether
Proline- imino group
is rigid and reduces
structural flexibility
Hydrophilic and can
form hydrogen
bonds with water;
sulfhydryl
polarity due to OH
or sulfhydryl group
or amide group; two
cysteines can be
oxidized to form a
covalently linked
(disulfide bond)
dimeric amino acid
called cystine.
Disulfide bonds are
very hydrophobic
Relatively
hydrophobic and
can participate in
hydrophobic
interactions.
OH group of
tyrosine can form
H bonds
N of tryptophan
indole ring makes
it more polar than
phenylalanine
All absorb UV
light at 280 nm
Hydrophilic due
to charge
Lysine has
positive charge
at pH7 due to the
second primary
amino group in
position
Arginine has a
positively
charged guanido
group Histidine has a imidazole group that is
ionizable at a pK near neutrality so can
act as a proton donor/acceptor
Hydrophilic; net negative charge at pH 7 due to carboxyl
group
Apart from the 20 common amino
acids, there are some 300 other that
have been found in cells
Key metabolites in biosynthesis of arginine and in the urea
cycle
Apart from the 20 common amino
acids, there are some 300 other that
have been found in cells
Plant cell
walls and
collagen
Zwitterion- dipolar ion that can act
as a H donor or acceptor
Dual
nature-
amphoteric;
ampholyte
Amino acid
titration
Note two
regions of
buffering power
pI is the
arithmetic
mean of the
pK values
Chemical environment perturbing
the expected pK values
The peptide bond- a substituted amide
linkage
-carboxyl -amino group acts as
Hydrolysis-
group a Condensation-
nucleophile to
exergonic but slow unfavorable,
displace the OH group
due to high carboxyl group must
activation energy be modified so that
OH can be lost
Dipeptide
Tetrapeptide- 4 residues
Free -amino and -carboxyl groups and the
nature and number of the ionizable R groups
can be used to predict the acid base behavior of
the peptide
The pKa of the R group changes if the amino
acid is a residue vs a free amino acid (as
expected due to the change in charges)
Like free amino acids, peptides have
characteristic titration curves and a
characteristic isoelectric point (pI) at which
they do not move in an electric field
Peptides
Naturally occurring peptides range in length from two
to many thousands of amino acid residues
Polypeptides generally have molecular weights below
10,000 while proteins have higher molecular weights
Even the smallest peptides can have biologically
important effects e.g. L-aspartyl-L-phenylalanine
methyl ester or some small peptide vertebrate
hormones or some antibiotics
Some hormones such as insulin (2 polypeptide chains,
one of 30 a acids and one of 21 a acids) are slightly
larger
So how long are the polypeptide chains?
Some proteins are multisubunit
If the subunits are identical then the protein is
oligomeric and the units are protomers e.g.
hemoglobin made of 2 chains and 2 chains
held by noncovalent interactions. Each paired
with a identically so it is a dimer of
protomers
Average molecular weights
Average molecular weight of the 20 amino
acids is about 138
Smaller amino acids predominate in most
proteins
Lose 1 molecule of H2O in bond formation (MW
18)
Taking the proportions into account, the
average molecular weight of protein amino
acids is 128
Average MW of a residue is about 110
Note that this is just a rough guide!!!
Amino acid
compositions
When completely hydrolysed,
each protein yields a
characteristic proportion of
amino acids, with some
occurring very infrequently
Acid hydrolysis changes
aspargine and glutamine to
aspartate and glutamate while
tryptophan is almost
completely degraded and
small amounts of serine,
threonine and tyrosine are lost
Proteins can contain other groups
Simple proteins have only amino acid
residues
Conjugated proteins contain permanently
associated chemical components as well
Non amino acid part is called its prosthetic
group
Prosthetic groups can be used to classify
proteins- e.g. lipoproteins, glycoproteins
and metalloproteins
Levels of protein structure
Most important aspect of primary structure is the
residue sequence. Primary Structure also includes a
description of the peptide and disulfide bonds
Secondary structure-stable arrangements giving rise
to recurring structural patterns
Tertiary structure describes
all aspects of the 3-D folding
Arrangement
of subunits in
space gives
quaternary
structure
Working with proteins
Need to purify proteins to
determine properties and
activities
Separation methods take
advantage of differing
properties such as size,
charge and binding
properties
First, break cells open to
make crude extract
If necessary, differential
centrifuge to prepare
subcellular fractions or
isolate organelles
Then fractionate using
differing properties
Ion exchange
chromatograph
y
Affinity
chromatograph
y
Size- exclusion
chromatography
Protein purification steps
HPLC- high performance liquid chromatography- use
high pressure to speed movement down column,
reduces transit time and reduces diffusional spreading
of bands improving resolution
Note that atseparates
Dialysis- each step,proteins
protein from
is lostsolvents,
and the uses
total a
starting amount decreases,
semipermeable membrane the total
with activity
pore size decreases
that retains
butproteins
the specific activity
but lets saltsincreases-
and buffersWhy?through- can only go
to equilibrium, so buffer changes necessary
PAGE and SDS-PAGE
Isoelectric focusing
pH gradient
established by
ampholytes
distributing in an
electric field
generated across
the gel. Protein
mixture is
applied and
proteins migrate
till it reaches the
pH that matches
its pI
Protein quantification
Use an assay system that can quantify specific
residues
If working with an enzyme, need to know the
reaction being catalyzed, a procedure for
determining the amount of substrate being
used up or product being formed, if other
factors are needed for the reaction, pH and
temperature
Unit of enzyme activity defined – 1 unit is the
amount of enzyme that causes the
transformation of 1 mol of substrate per
minute at 25ºC under optimal conditions
Amino acid sequence determines how a
protein folds to give it a unique 3-D shape
and this determines its function
Amino acid sequence of a protein is not
always fixes, and polymorphisms exist
Some polymorphisms do not affect
biological function while others can be
debilitating or lethal
What do identical sequence residues
from different proteins tell us about
the proteins?
More alignments- common ancestry
relationships between EF-1 and EF-TU
Homologs-members of protein families
Paralogs-two proteins of the same family
present in the same species
Orthologs- two proteins of the same family
in different species
Bacterial evolutionary tree based on
the GroEL family of proteins
