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The highlights are:
• Amino acids
• Peptides and proteins
• Working with proteins
• The structure of proteins: primary structure
1.
Chap. 3A Amino Acids, Peptides, and
Proteins
• Amino acids
• Peptides and proteins
• Working with proteins
• The structure of proteins:
primary structure
Fig. 3-6. Absorption of ultraviolet
light by aromatic amino acids.
2.
Introduction to Proteins
Proteins mediate nearly every process that takes place inside a
cell. They are the most abundant biological macromolecules in
cells. All proteins, regardless of organism, are composed of the
same set of 20 amino acids that are incorporated into them during
translation. Due to the nearly limitless variety in the sequences of
amino acids in proteins, nearly all imaginable functions can be
encoded in proteins (Fig. 3-1).
3.
Common Features of Amino Acids
There are 20 "standard" amino acids that are
specified by the genetic code and polymerized
into proteins by ribosomal translation. All
amino acids contain an carbon to which
typically 4 different substituent groups are
attached (Fig. 3-2). These groups are the -
amino group, the -carboxyl group, hydrogen,
and the variable R group (side-chain). (Note
that in glycine, the R group consists of
another hydrogen atom. In the other 19
amino acids the carbon is a chiral center.)
The -amino and -carboxyl groups are
charged at neutral pH. There are two possible
configurations for these four substituents--
the "D" and "L" stereoisomers, which are
mirror images of each other (enantiomers)
(Fig. 3-3). The standard amino acids have the
L-configuration. Amino acids are classified
based on the characteristics of their R
groups. The chemical properties of the
standard 20 amino acids are summarized in
Table 3-1 (next two slides).
6.
Numbering of Carbons in Amino Acids
The conventions for labeling the carbon atoms in amino acids is
illustrated using lysine in the figure. The carbon is always
carbon-2 of the amino acid. The -carboxyl group is always
7.
D,L Classification System for Amino Acid
Configurations
The D, L system is used to specify the absolute configuration of
substituent groups about chiral carbons in amino acids and
monosaccharides. In this system, amino acids are specified as D
or L based on comparison of their configurations to the
reference compounds, D- and L-glyceraldehyde (Fig. 3-4). Note
that not all amino acids actually are levorotatory. Thus D and L
designate only the configurations of the substituent groups and
not the optical properties of the amino acid.
8.
Classification of Amino Acids by R Group
Amino acids are collected into different categories based on
similarities in the properties of their R groups. One such
classification scheme (Fig. 3-5) relies heavily on the polarity
of the R groups. Note that some amino acids such as glycine,
histidine, and cysteine do not fit perfectly into any one group.
Thus, their assignments to a particular group are somewhat
subjective rather than based on absolute characteristics. The
textbook places the amino acids into five categories based on
the properties of their R groups--nonpolar, aliphatic;
aromatic; polar, uncharged; positively charged; and negatively
charged (next slides).
9.
Nonpolar, Aliphatic Amino Acids
The amino acids in this group lack polar functional groups in their
side chains. Due to the hydrophobicity of their R groups, they
often cluster together within the interior of proteins, stabilizing
protein structure via hydrophobic interactions. The preferences of
several of these amino acids for regions of protein secondary
structure are discussed in Chap. 4.
10.
Aromatic Amino Acids
The side-chains of the aromatic amino acids, phenylalanine,
tyrosine, and tryptophan, overall are very hydrophobic. The R
group of tyrosine also contains a polar hydroxyl group that can
participate in H bonding interactions. The R groups of tyrosine,
and particularly tryptophan, absorb ultraviolet light at a
maximum of 280 nm wavelength (Fig. 3-6). Light absorption by
these amino acids is exploited in detecting and quantifying
proteins in the laboratory using the technique of absorbance
11.
Polar, Uncharged Amino Acids
The R groups of the polar, uncharged
amino acids all contain polar
functional groups that can hydrogen
bond with water. Serine and
threonine contain hydroxyl groups,
asparagine and glutamine contain
amide groups, and cysteine contains a
sulfhydryl group, albeit whose
polarity is quite weak. Asparagine and
glutamine are the amide forms of the
two negatively charged amino acids
aspartate and glutamate. The
sulfhydryl group of the cysteine side
chain is a weak acid (pKa = 8.2). The
cysteine side chain therefore is
mostly uncharged at neutral pH.
12.
Cysteine and Disulfide Bonds
The thiol groups of two cysteine residues are readily oxidized
to form a covalently linked dimeric amino acid known as cystine.
In cystine, the two cysteines are joined by a disulfide bond
(Fig. 3-7). The disulfide-linked cystine residue is strongly
hydrophobic. In proteins, disulfide bonds form covalent links
between different parts of a polypeptide chain, or between two
different polypeptide chains.
13.
Positively Charged Amino Acids
The most hydrophilic R groups are those that are either
positively or negatively charged. The side-chains of lysine and
arginine are fully positively charged at neutral pH. In lysine, a
primary amino group is attached to the carbon of the side-
chain. In arginine, the guanidinium group of the side-chain is
postively charged. The histidine R group contains an aromatic
imidazole group that is partially positively charged at neutral pH
(Refer to Worked Example 2-5). Histidine residues function in
many enzyme-catalyzed reactions as proton donors and/or
14.
Negatively Charged Amino Acids
The R groups of aspartate and glutamate contain carboxyl
groups that are fully negatively charged at neutral pH (pKRs of
3.65 and 4.25, respectively, Table 3-1). In aspartate, the
carboxyl group is attached to the ß carbon of the amino acid
backbone. In glutamate, the carboxyl group is attached to the
15.
Intro. to Amino Acid Ionization (I)
The -carboxyl and -amino groups of all amino acids, along with
the ionizable R groups of 7 amino acids, function as weak acids
and bases in aqueous solutions (Table 3-1). The pKas of these
functional groups depend on the chemical properties of the groups
themselves and range between 1.8-2.4 for the -carboxyl groups,
and 8.8-11.0 for -amino groups. When a simple amino acid such
as alanine, which lacks an ionizable functional group in its side-
chain, is dissolved in water at neutral pH, its -carboxyl group is
negatively charged, and its -amino group is positively charged.
Such dipolar ions (total charge equals 0) are called zwitterions.
Substances having this dual (acid-base) nature are amphoteric and
are often called ampholytes. The ionization behavior of the -
carboxyl and -amino groups of a simple amino acid such as
alanine are shown in the diagram below.
16.
Intro. to Amino Acid Ionization (II)
The nonionic and zwitterionic forms of a simple amino acid such
as alanine are shown in Fig. 3-9. The zwitterionic form
predominates at neutral pH. The nonionic form does not occur in
significant amounts in aqueous solution at any pH. A zwitterion
can act as either an acid (proton donor) or a base (proton
17.
Titration of Simple Amino Acids
The titration curves of simple amino acids
such as glycine (Fig. 3-10) that have non-
dissociable R groups, have two plateaus,
which correspond to the dissociation and
titration of the -carboxyl group (pK1,
left) and the -amino group (pK2, right).
As shown above the curve, the
predominant ionic species in solution at low
pH is the fully protonated form, +H3N-
CH2-COOH (net charge = +1), In between
the two plateaus, the zwitterionic form,
H3N-CH2-COO- (net charge = 0)
predominates. At the end of the titration,
the fully dissociated species H2N-CH2-
COO- (net charge -1) predominates. The
curve shows that glycine has two regions
of buffering
above pK1 andpower centered
pK2. The ±1 pH unit
Henderson-Hasselbalch equation can be
used to calculate the amounts of the conjugate acid and
conjugate base species in solution at any pH. Lastly, the pH at
which the zwitterionic (0-charged) species of glycine
predominates (one equivalent of OH- added) is called the
isoelectric point or isoelectric pH. The isoelectric pH is exactly
halfway between the two pKas for glycine.
18.
Chemical Environment and the pKa
The pKas of the -carboxyl groups of all amino acids are lower
than the pKas of the carboxyl groups in methyl-substituted
carboxylic acids such as acetic acid (Fig. 3-11). This is due to the
local chemical environment of the -carboxyl groups in amino acids.
Namely, placement near the -amino group, which is positively
charged, makes the -carboxyl groups of amino acids more acidic
than the carboxyl group of acetic acid. Similarly, the chemical
environment near -amino groups makes them more acidic than the
amino groups of a methyl-substituted amino compounds such as
methylamine. In this case the electron withdrawing properties of
the oxygens on the -carboxyl groups of amino acids make the -
amino groups hold onto their protons less tightly than in other
19.
Titration of Glutamate
The acidic amino acid, glutamate,
has a second carboxyl group present
in its side-chain. Thus the titration
curve for glutamate (and aspartate)
has three plateaus, each one
corresponding to the dissociation of
a proton from the amino acid (Fig.
3-12a). Since the R group carboxyl
group has a pKR between that of the
pK1 and pK2, the second plateau
corresponds to the titration of this
group. Based on inspection of the
ionic forms in solution (top) it is
clear that the zwitterionic form of
glutamate occurs at a pH midway
between that of pK1 and pKR. Thus
the pI for glutamate is 3.22.
20.
Titration of Histidine
Histidine has an imidazole R group
that contains a dissociable proton
with a pKR of 6.0. Thus the
titration curve for histidine also
has three plateaus (Fig. 3-12b).
For histidine, the 0-charged
zwitterionic species occurs in
solution at a pH midway between
pKR and pK2 (pI = 7.59). Because
the histidine pKR is near
neutrality, the R group of
histidine plays a role in buffering
the pH of solutions containing
21.
Peptide Bonds
Peptide bonds are amide linkages that join amino acids in
oligopeptides, polypeptides, and proteins. Peptide bonds are
formed by condensation reactions in which the elements of water
are removed (dehydration) from the reacting -amino and -
carboxyl groups that come together to form the bond (Fig. 3-
13). Note that peptide bonds in proteins are formed in vivo via a
different mechanism than the one shown in the diagram. The
term oligopeptide refers to polymers with relatively few amino
acid residues. The term polypeptide signifies polymers of
generally less than 10,000 mw, whereas the term protein refers
to longer polymers.
22.
Structure of (Oligo)peptides
The structure of the pentapeptide, serylglycyltyrosylalanylleucine
(Ser-Gly-Tyr-Ala-Leu, SGYAL) is shown in Fig. 3-14. Note that
all five amino acids are linked by peptide bonds (shaded groups).
R groups are shown in red. When an amino acid sequence of a
peptide, polypeptide, or protein is shown, by convention the
amino-terminal (N-terminal) end is placed on the left, and the
carboxyl-terminal (C-terminal) end is place on the right. The
amino acid sequence is read left-to-right beginning with the N-
terminal end.
23.
Ionization Behavior of Peptides
The -amino and -carboxyl groups of
amino acid residues that are joined
together in peptide bonds do not
undergo ionization reactions near
physiological pH (Fig. 3-15). The free
-amino group at the N-terminus of
the peptide, and the free -carboxyl
group at the C-terminus ionize as they
do in isolated amino acids. Likewise the
R groups that contain ionizable
functional groups continue to ionize as
in free amino acids. Note, however,
that the pKa values for ionizable
residues in peptides change somewhat
from their values in free amino acids
due to changes in their local chemical
environments. Like free amino acids,
peptides have characteristic titration
curves and an isoelectric point at which
the net charge on the peptide is zero.
These properties are exploited in some
of the separation techniques discussed
later in the chapter.
24.
Aspartame
Naturally occurring peptides range in length from two to many
thousands of amino acid residues. Even small peptides can be
biologically active. For example, the commercially synthesized
dipeptide L-aspartyl-L-phenylalanine methyl ester is the sweetener
called aspartame or NutraSweet. Other examples of short
biologically active peptides are thyrotropin-releasing factor (three
amino acid residues) and oxytocin (nine amino acid residues).
25.
Protein Sizes
The lengths of polypeptide chains in proteins vary considerably
(Table 3-2). While the great majority of proteins contain fewer
than 2,000 amino acids, some are much larger. The largest known
protein is titin (26,926 amino acids), which is a component of
vertebrate muscle. Some proteins consist of a single polypeptide
chain, whereas others called multisubunit proteins have two or
more associated polypeptides. The individual polypeptide chains in
a multisubunit protein can be identical or different. If at least
two subunits are identical, the protein is said to be oligomeric
and the identical units (consisting of one or more polypeptide
chains) are referred to as protomers.
26.
Amino Acid Compositions of Proteins
The amino acid compositions of proteins differ between proteins
and are highly variable (Table 3-3). Some amino acids occur only
once or not at all in a given protein and other amino acids may
occur in large numbers.
Based on the frequencies
at which the 20 standard
amino acids occur in
proteins, and their
molecular weights (Table
3-1), the average
molecular weight of an
amino acid in a protein is
128. Because a molecule
of water (Mr 18) is lost on
creation of each peptide
bond, the average
molecular weight of an
amino acid residue in a
protein is about 110.
27.
Conjugated Proteins
Simple proteins, such as chymotrypsin, contain only amino acids.
Other proteins--conjugated proteins--contain other associated
chemical components in addition to amino acids. The non-amino
acid part of a conjugated protein is called its prosthetic group.
Examples of conjugated proteins and their prosthetic groups are
listed in Table 3-4.
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