Chap. 3B Amino Acids, Peptides, and Proteins

1
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.
2
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.
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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.
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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.
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Worked Example 3-1. Ion Exchange of Peptides
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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 25C 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).
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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.
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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.
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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.
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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 Sangers
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.
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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).
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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).
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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.
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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).
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Representations of Consensus Sequences
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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.
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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.