Chap.

3B Amino Acids, Peptides, and

Proteins
Topics
• 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.
 Overview of Protein Purification
To study a protein in detail, a researcher must be able to separate
it from other proteins in pure form and must have the techniques
to determine its properties. To purify a protein, one usually starts
with a crude extract of a tissue or cell sample and separates the
proteins within it into fractions. Given that the initial volume of the
crude extract is relatively large a researcher typically applies a
technique like ammonium sulfate precipitation to reduce the size of
the sample and the number of proteins within it. With ammonium
sulfate precipitation, one exploits differences in the solubility of
proteins in salt solution to obtain a fraction that is enriched in the
protein of interest. Throughout the multiple steps of protein
purification, the researcher must have available some type of assay
for monitoring the presence of the protein. Subsequent to
ammonium sulfate precipitation, investigators typically apply column
chromatography procedures to further purify the protein.
 Column Chromatography
Column chromatography procedures
can separate proteins based on their
net charge at a given pH (ion-
exchange chromatography), their
relative sizes (size-exclusion
chromatography), and their ligand
binding specificity (affinity
chromatography). The general
principle behind column
chromatography procedures is
illustrated in Fig. 3-16. As
different proteins percolate through
the column they are separated
based on their physical properties.
The effluent fraction containing the
protein of interest can be identified
based on an enzymatic or other type
of assay.
 Ion-exchange Chromatography
In ion-exchange chromatography, proteins are separated based
on differences in the sign and magnitude of their charges at a
given pH (Fig. 3-17a). So-called cation exchange resins are used
to fractionate positively charged proteins in a mixture. These
resins contain bound negative functional groups (inset).
Anion exchange resins are used to
fractionate negatively charged
proteins in a mixture. Anion
exchange resins contain bound
positive functional groups. With
both techniques proteins having
the same net charge as the resin
move through the column relatively
quickly. Proteins with a net charge
that is opposite to that of the
resin are retained, and ultimately
are released by adjusting the pH
or salt concentration of the elution
buffer.
Worked Example 3-1. Ion Exchange of
Peptides
 Size-exclusion Chromatography
In size-exclusion chromatography (gel
filtration) proteins are separated
based on their size (molecular weight)
(Fig. 3-17b). The resin used in size-
exclusion chromatography is uncharged,
but contains pores into which small
solutes/proteins may be able to
penetrate. For this reason, the largest
proteins move through the column the
fastest, whereas small proteins/salts
are retained longer. Size exclusion
chromatography in the presence of
standards of known Mr can be used to
determine the approximate molecular
weight of an unknown protein.
 Affinity Chromatography
In affinity chromatography, the
resin in the column contains a
covalently attached chemical
group called a ligand that is
bound by the protein of interest
(Fig. 3-17c). Thus when a
mixture of proteins containing
the protein that recognizes the
ligand is applied to the column,
other proteins pass through in a
wash of the column, while the
protein of interest is retained.
The protein of interest
ultimately is eluted from the
column by adding a buffer
containing the free ligand. The
free ligand competes with the
bound ligand for binding to the
protein of interest, and the
protein dissociates from the
resin. Very often, affinity
chromatography gives the largest
fold-purification of any step
used in protein purification.
High-performance Liquid Chromatography
In high-performance liquid chromatography (HPLC), the elution
buffer is pumped over the resin at high pressure and speed.
This reduces the transit time of the protein on the column and
improves the resolution of separation by reducing diffusional
spreading of protein bands during elution.
 Purification Tables
The progress of purification of a protein of interest is recorded
in a purification table (Table 3-5). With each step, the mass of
total protein recovered is reduced while the specific activity
(units/mg) of the protein fraction becomes greater.
 SDS PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS
PAGE) is a routinely applied analytical method to estimate the
purity and molecular weight of proteins in a sample (Fig. 3-18).
Proteins are dissolved in a buffer containing SDS and separated
on a polyacrylamide gel matrix by the application of an electrical
field (see next slide for mechanism of separation). The locations
of proteins on the gel after electrophoretic separation is
determined by staining with a protein reactive dye such as
Coomassie blue dye. The figure shows an application where SDS
PAGE was used to monitor the status of purification of the RecA
protein of E. coli.
 Mr Estimation by SDS PAGE
In SDS PAGE, proteins are uniformly coated by SDS with about
one molecule of SDS being bound per amino acid residue. Since
SDS is strongly negatively charged under the electrophoresis
buffer conditions used, all proteins adopt a roughly equal charge
to mass ratio. They further all adopt a rod-shaped structure in
solution. Thus separation occurs based solely on Mr, with smaller
proteins moving faster than larger proteins through the sieving
matrix of the gel. The Mr of an unknown protein can be
determined by running it on the gel in parallel with known Mr
standards (Fig. 3-19).
 Isoelectric Focusing
In isoelectric focusing, proteins are separated according to their
isoelectric points (pIs) (Fig. 3-20). A pH gradient is established
on a gel strip by allowing a mixture of low molecular weight
organic acids and bases (ampholytes) to distribute themselves in
an electric field applied across the strip. Proteins migrate on the
strip to a point where the pH matches their pIs. At the pI the
net charge on a protein is zero, and it stops moving in the
electric field.
 Two-dimensional Gel Electrophoresis
In two-dimensional gel electrophoresis,
isoelectric focusing and SDS PAGE are
combined to obtain a high resolution
separation of a complex mixture of
proteins (Fig. 3-21). Proteins are first
separated by isoelectric focusing on a
gel strip. Then the gel strip is mounted
on top of an SDS PAGE gel and
separated by electrophoresis in the
second dimension. Horizontal separation
of spots is based on their pI
differences. Vertical separation is
based on differences in molecular
weights. Thousands of proteins can be
separated by this techniques using a
single SDS PAGE gel. Individual protein
spots can be excised from the gel and
identified by mass spectrometry.
 Activity vs Specific Activity
The purification of an enzyme is
measured, or assayed, based on the
ability of the protein fraction containing
the enzyme to carry out a biochemical
reaction. 1.0 unit of enzyme activity is
defined as the amount of enzyme
causing the transformation of 1.0 mol
of substrate to product per minute at
25˚C under optimal conditions of
measurement. The term activity reflects
the total units of enzyme in a solution.
The term specific activity is the number
of units of enzyme activity per milligram
of total protein (Fig. 3-22). The
specific activity is a measure of enzyme
purity. It increases during the
purification of an enzyme and become
maximal and constant when the enzyme
is pure (see Table 3-5).
 Levels of Structure in Proteins
The structure of large molecules such as proteins can be
described at four levels of complexity, arranged in a conceptual
hierarchy (Fig. 3-23). The primary structure of a protein refers
to the sequence of amino acids in the protein, and includes
disulfide bonds. The secondary structure refers to local and
stable folding elements in the larger structure, such as  helices.
The tertiary structure refers to the three-dimensional folding and
locations of all atoms in the protein. Quaternary structure is
reserved for multisubunit proteins. The arrangement in 3D space
of all subunits in a protein is its quaternary structure.
 Amino Acid Sequence of Bovine Insulin
The first protein whose sequence was determined was that of
bovine insulin (Fig. 3-24). The British Nobel laureate, Frederick
Sanger, oversaw the sequencing of bovine insulin in 1953, the same
year that Watson and Crick solved the structure of double-helical
DNA. Today, few protein sequences are determined in their
entirety by chemical methods, with most being deduced from the
DNA sequences of the genes that encode them. However, segments
of proteins are often sequenced in the process of gene cloning, and
many of the classical steps used in protein sequencing are applied
today in the study of protein structure and function. In the next
few slides modern methods for protein sequencing by chemical
procedures and mass spectrometry are discussed.
 Overview of Protein Sequencing
The conventional strategy used for
chemical sequencing of a protein is
summarized in Fig. 3-25. It
includes 1) identification of the
first residue located at the amino
terminus, 2) determination of the
complete amino acid composition of
the protein, 3) cleavage of the
protein into shorter polypeptide
fragments that can be sequenced in
their entirety, 4) sequencing of
each polypeptide fragment, and 5)
ordering the polypeptide fragments
in the overall sequence of the
protein. Each of these steps is
covered further in the next few
slides.
 N-terminal Labeling Reagents
The first amino acid residue at the N-terminus of a protein is
identified by reaction of the protein with one of the reagents such
as FDNB (1-fluoro-2,4-dinitrobenzene) shown in Fig. 3-26.
Following labeling the protein is hydrolyzed and the modified N-
terminal residue is identified by chromatography. FDNB is also
known as Sanger’s reagent. Other more sensitive fluorescent
reagents (e.g., dansyl chloride and dabsyl chloride) now are used
when working with small quantities of a purified protein. N-
terminal labeling also identifies which sequenced polypeptide occurs
first in the complete sequence of the protein, as illustrated in Fig.
3-25.
 Edman Degradation Sequencing
The chemical sequencing process itself is based on a two-step
procedure developed by Pehr Edman (Fig. 3-27). The reagent
phenylisothiocyanate is used to label the N-terminal residue of
the polypeptide, which then is released and identified without
damaging the remainder of the polypeptide. Then the process is
repeated. About 40 amino acids can be sequenced for each
polypeptide. Polypeptides themselves are generated by enzymatic
or chemical fragmentation of the starting protein (Fig. 3-25).
 Breaking Disulfide Bonds in Proteins
In order to obtain isolated polypeptide fragments from a protein
for sequencing, disulfide bonds must be broken. Commonly bonds
are broken by oxidation with performic acid, or using a two-step
procedure in which the bond is first broken by treatment with a
reducing agent such as dithiothreitol, and then the free thiols
are alkylated by carboxymethylation using iodoacetate (Fig. 3-
28).
 Methods for Fragmenting Proteins
Since at most 40 amino acids at a time can be sequenced by the
Edman degradation procedure, large proteins must be fragmented
into smaller polypeptides for sequencing. A number of proteases
are available for this purpose as are a few chemical cleavage
methods (e.g., cyanogen bromide cleavage) (Table 3-6). The
selection of a method for cleavage is guided in part by the earlier
determination of the total amino acid composition of the protein.
The identification of the C-terminal residue in the polypeptide
fragments helps in ordering the polypeptides in the overall
sequence (see Fig. 3-25). The ordering of the protein sequence
also is facilitated by sequencing in parallel a second set of
polypeptides generated by another protease. The order in which
the fragments appeared in the original protein can then be
determined by examining the overlaps in sequence between the two
sets of fragments.
Information Derived from Protein Sequence
Analysis
The analysis of protein sequences via computer-based tools
(bioinformatics) provides a wealth of information about the
structure and function of proteins, and the evolutionary
relationships between the organisms that synthesize them. A key
method in the application of bioinformatics to evolutionary,
structure and function analysis are sequence alignments (Fig. 3-
33). Sequence alignments of related proteins also reveal
consensus sequences that indicate protein function, cell location,
chemical modification and prosthetic group binding, and even
turnover rates (Box 3-2, next slide).
Representations of Consensus Sequences
 Bacterial Evolutionary Trees
Evolutionary relationships between organisms are revealed by
comparing the sequences of fundamental proteins that are present
in all organisms. A bacterial evolutionary tree derived from
comparison of the sequences of the protein GroEL (a chaperone
involved in assisted protein folding) is shown in Fig. 3-35. In such
trees, external nodes mark the locations of extant organisms.
Internal nodes mark the locations of their last common ancestors.
The lengths of lines represent the level of sequence divergence
between organisms. Note that comparisons of protein sequence
are more reliable than comparisons of DNA sequences for tree
construction, since proteins contain 20 amino acids and DNA
contains only 4 bases.
 Consensus Tree of Life
Sequence comparisons of numerous shared proteins and analysis
of additional genomic features has been performed to construct
the mostly likely general tree of life (Fig. 3-36). Such analysis
(and earlier rRNA sequencing by Carl Woese) reveal that all life
forms on Earth can be divided into three domains--Bacteria,
Archaea, and Eukaryotes. The evolutionary distances of each
kingdom to the last universal common ancestor (LUCA) are given
by the lengths of the lines in the tree.