Chapter 3 Flashcards
stupid textbook isnt working
oh no
Substances having this dual (acid-base) nature are amphoteric and are o
led ampholytes
(from “amphoteric electrolytes”). A simple monoamino monocarboxylic α-amino acid, such as
alanine, is a diprotic acid when fully protonated; it has two groups, the —COOH group and the
—NH
+
3 group, that can yield protons
good pics on pg 373
kk
The 20 amino acids commonly found as residues in proteins contain an \and thus amino acids can exist in at least two
stereoisomeric forms.
378
Only the L stereoisomers of amino acids, with a configuration related to the absolute
configuration of the reference molecule L-glyceraldehyde, are found in proteins.
Amino acids can be classified into five types on the basis of the polarity and charge (at pH 7) of
their R groups.
Other, less common amino acids also occur, either as constituents of proteins (usually through
modification of common amino acid residues a
α-carboxyl group, an αamino group, and a distinctive R group substituted on the α-carbon atom.
The α-carbon atom of
all amino acids except glycine is asymmetric,
\and thus amino acids can exist in at least two
stereoisomeric forms.
Only the L stereoisomers of amino acids, with a configuration related to the absolute
configuration of the reference molecule L-glyceraldehyde, are found in
proteins
Amino acids can be classified into five types on the basis of
the polarity and charge (at pH 7) of
their R groups.
Other, less common amino acids also occur, either as
s constituents of proteins (usually through
modification of common amino acid residues a
Amino acids vary in their acid-base properties and have characteristic
titration curves.
Monoamino monocarboxylic amino acids (with nonionizable R groups) are diprotic acids
(+H3NCH(R)COOH) at low pH and exist in several different ionic forms as the pH is increase
Amino acids with ionizable R groups have additional
ionic species, depending on the pH of the
medium and the pKa of the R group
Ionizable R groups in a peptide (Table 3-1) also
contribute to
the overall acid-base properties of the molecule
Many small peptides exert their effects at
very low
concentrations. For example, a number of vertebrate hormones
(Chapter 23) are small peptides. These include oxytocin (nine
amino acid residues), which is secreted by the posterior pituitary
gland and stimulates uterine contractions, and thyrotropinreleasing factor (three residues), which is formed in the
hypothalamus and stimulates the release of another hormone,
thyrotropin, from the anterior pituitary gland. Some extremely
toxic mushroom poisons, such as amanitin, are also small
peptides, as are many antibiotics.
Amino acids can be
joined covalently through peptide bonds to
form peptides and proteins. Cells generally contain thousands of
different proteins, each with a different biological activity.
The ionization behavior of peptides reflects their ionizable side
chains as well as
the terminal α-amino and α-carboxyl groups.
Proteins can be very long polypeptide chains of
100 to several
thousand amino acid residues. However, some naturally
occurring peptides have only a few amino acid residues. Some
proteins are composed of several noncovalently associated
polypeptide chains, called subunits.
Simple proteins yield only amino acids on
hydrolysis;
conjugated proteins contain in addition some other component,
such as a metal or organic prosthetic group.
Ion-exchange chromatography
exploits differences in the sign
and magnitude of the net electric charge of proteins at a given pH
Affinity chromatography
is based on binding affinity (Fig. 3-17c).
The beads in the column have a covalently attached chemical
group called a ligand — a group or molecule that binds to a
macromolecule such as a protein.
Protein purification protocols o
fuse additional amino acids or peptides (tags) to the target
protein. Affinity chromatography can be used to bind this tag,
achieving a large increase in purity in a single step (see Fig. 9-11).
In many cases, the tag can be subsequently removed, fully
restoring the function of the native protein.
electrophoresis
Protein purification is usually complemented by electrophoresis,
an analytical process that allows researchers to visualize and
characterize proteins as they are purified.
The electrophoretic method commonly employed for estimation
of purity and molecular weight makes use of the detergent
sodium dodecyl sulfate (SDS) (“dodecyl” denoting a 12-carbon
chain).
Isoelectric focusing
is a procedure used to determine the
isoelectric point (pI) of a protein (Fig. 3-20).
A pH gradient is
established by
y allowing a mixture of low molecular weight
organic acids and bases (ampholytes; p. 77) to distribute
407
themselves in an electric field generated across the gel. When a
protein mixture is applied, each protein migrates until it reaches
the pH that matches its pI. Proteins with different isoelectric
points are thus distributed differently throughout the gel.
Combining isoelectric focusing and SDS electrophoresis
sequentially in a process called
two-dimensional electrophoresis
permits the resolution of complex mixtures of proteins (Fig. 3-
21). This is a more sensitive analytical method than either
electrophoretic method alone. Two-dimensional electrophoresis
408
separates proteins of identical molecular weight that differ in pI,
or proteins with similar pI values but different molecular weights.
Proteins are separated and purified on the basis of
f differences
in their properties. Proteins can be selectively precipitated by
changes in pH or temperature, and particularly by the addition of
certain salts. A wide range of chromatographic procedures makes
use of differences in size, binding affinities, charge, and other
properties. These include ion-exchange, size-exclusion, affinity,
and high-performance liquid chromatograph
Electrophoresis separates proteins on the basis of
mass or
charge for analytical purposes. SDS gel electrophoresis and
isoelectric focusing can be used separately or in combination for
higher resolution.
All purification procedures require
e a method for quantifying or
assaying the protein of interest in the presence of other proteins.
Purification can be monitored by assaying specific activity
primary structure
A description of all covalent bonds (mainly
peptide bonds and disulfide bonds) linking amino acid residues in
a polypeptide chain is its primary structure. The most important
element of primary structure is the sequence of amino acid
residues.
secondary structure
refers to particularly stable
arrangements of amino acid residues giving rise to recurring
structural patterns.
tertiary structure
describes all aspects of the
three-dimensional folding of a polypeptide.
quaternary structure
When a protein has
two or more polypeptide subunits, their arrangement in space is
referred to as quaternary structure. Our exploration of proteins
will eventually include complex protein machines consisting of
dozens to thousands of subunits
good pic on p 417
kk
what does it mean for fxn if a protein has a unique amino acid sequence
this confers a particular threedimensional structure. This structure in turn confers a unique
function.
Enzymes called proteases
catalyze the hydrolytic cleavage of
peptide bonds and provide the most common method to break a
protein into parts. Some proteases cleave only the peptide bond
adjacent to particular amino acid residues (Table 3-6) and thus
fragment a polypeptide chain in a predictable and reproducible
way. A
. A few chemical reagents also
cleave the peptide bond
adjacent to specific residues. Among proteases, the digestive
enzyme trypsin catalyzes the hydrolysis of only those peptide
bonds in which the carbonyl group is contributed by either a Lys
or an Arg residue, regardless of the length or amino acid
sequence of the chain.
Mass spectrometry can
provide a highly accurate measure of the
molecular mass of a protein, readily distinguishing between
single proton differences. it can also do much more
what more can mass spectrometry allow for
The sequences of multiple short polypeptide segments (20
426
to 30 amino acid residues each) in a protein sample can be
obtained within seconds.
Electrospray ionization mass spectrometry of a protein (this is a pic on p 429)
A
protein solution is dispersed into highly charged droplets by passage
through a needle under the influence of a high-voltage electric field. The
droplets evaporate, and the ions (with added protons in this case) enter the
mass spectrometer for m/z measurement. (b) The spectrum generated is a
family of peaks, with each successive peak (from right to le
The analysis of complex mixtures of proteins — even entire
cellular proteomes — is facilitated by
y liquid chromatography (LC)
that is integrated into the instrument (LC-MS/MS). The organism
of interest is generally one in which the genomic sequence is
known. Cellular proteins are first isolated in an extract, then
digested into relatively short peptides by a protease such as
trypsin. The very complex mixture of peptides is subjected to
chromatography, so that resolved peptides are introduced to the
mass spectrometer successively
Many peptides are potentially useful as pharmacologic agents,
and their production is of considerable commercial importance.
In addition to its commercial applications, the synthesis of
specific peptide portions of larger proteins is an increasingly
important tool for the study of protein structure and function.
There are three ways to obtain a peptide:
(1) purification from
tissue, a tasok
oomade difficult by the vanishingly low
concentrations of some peptides; (2 (2) genetic engineering
(Chapter 9); and (3) direct chemical synthesis. Powerful
techniques now make direct chemical synthesis an attractive
option in many cases.
The complexity of proteins makes the traditional synthetic
approaches of organic chemistry impractical for peptides with
more than four or five amino acid residues. One problem is
the
difficulty of purifying the product after each step
Knowledge of the sequence of amino acids in a protein can
offer insights into
its three-dimensional structure and its
function, cellular location, and evolution. Most of these insights
are derived by searching for similarities between a protein of
interest and previously studied proteins. Comparison of a newly
obtained sequence with sequence data in international
repositories often reveals relationships both surprising and enlightening.
Much of the functional information encapsulated in protein
sequences comes in the form of consensus sequences. This term
is applied to
such sequences in DNA, RNA, or protein. When a
series of related nucleic acid sequences or protein sequences are
compared, a consensus sequence is the one that reflects the most
common base or amino acid at each position.
horizontal gene transfer
Another complicating factor in tracing evolutionary history is the
rare transfer of a gene or a group of genes from one organism to
another, a process called horizontal gene transfer.
Differences in protein function result from
differences in
amino acid composition and sequence. The chemical properties
of particular amino acid residues are often critical to the function
of a protein.
Most amino acid sequences are deduced from
genomic
sequences and by mass spectrometry. Methods derived from
classical approaches to protein sequencing remain important in
protein chemistry
Short proteins and peptides (up to about 100 residues) can be
chemically synthesized. The peptide is built up, one amino acid
residue at a time, while tethered to a solid support
Protein sequences are a rich source of
information about
protein structure and function. Bioinformatics can analyze
changes in the amino acid sequences of homologous proteins
over time to trace the evolution of life on Earth.
