• Protein purification table

• Protein amino acid sequencing methods

• Introduction about structure of proteins
The Function of a Protein Depends on Its Amino Acid Sequence

• The Amino Acid Sequences of Millions of Proteins Have Been Determined

• Large Proteins Must Be Sequenced in Smaller Segments
• The overall accuracy of amino acid sequencing generally declines as the length of the
polypeptide increases. The very large polypeptides found in proteins must be broken
down into smaller pieces to be sequenced efficiently.
• There are several steps in this process. First, the protein is cleaved into a set of specific
fragments by chemical or enzymatic methods. If any disulfide bonds are present, they
must be broken.
 Proteins can be separated by various
chromatographic methods:
Ion exchange, size exclusion and affinity
chromatography methods

By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0
micro mol of substrate per minute at 25 C under optimal conditions of measurement. The term activity refers to the total
units of enzyme in a solution.
The specific activity is the number of enzyme units per milligram of total protein . The specific activity is a measure of
enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure
Exercise

Find out specific activity from above data

 A Purification Table for a Hypothetical Enzyme
• The term activity refers to the total units of enzyme in a solution.
• The specific activity is the number of enzyme units per milligram of total
protein.
• The specific activity is a measure of enzyme purity: it increases during
purification of an enzyme and becomes maximal when the enzyme is pure
• After each purification step, the activity of the preparation is assayed, the
total amount of protein is determined, and the ratio of the two gives the
specific activity.
• Activity and total protein generally decrease with each step.
• Activity decreases because some loss always occurs due to inactivation or
non-specific interactions with column material or other molecules in the
Activity versus specific activity. The
difference between these terms can be solution.
illustrated by considering two flasks containing
marbles. The flasks contain the same number • Total protein decreases because unwanted or nonspecific protein are removed.
of red marbles, but different numbers of
marbles of other colors. If the marbles • A protein is generally considered pure when further purification steps fail to
represent proteins, both flasks contain the same
activity of the protein represented by the red increase specific activity and when only a single protein species can be
marbles. The second flask, however, has the
higher specific activity because red marbles detected.
represent a higher fraction of the total.

1 U (μmol/min) of an enzyme is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the conditions of the assay method
Proteins Can Be Separated and Characterized by Electrophoresis

Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the SDS
polyacrylamide gel. The proteins move into the gel when an electric field is applied.
(b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie
blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different
protein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and
therefore are found nearer the bottom of the gel.
2-dimensional electrophoresis: Isoelectric focusing and SDS electrophoresis

A pH gradient is established by allowing a

mixture of low molecular weight organic acids
and bases (ampholytes) to distributed 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.
Two-dimensional electrophoresis separates
proteins of identical molecular weight that
differ in pI, or proteins with similar pI values
but different molecular weights.
Protein sequencing methods a. Direct protein sequencing procedure were developed by Fred Sanger to
sequence insulin and have since been used for many additional proteins. FDNB (1-
fluoro-2,4-dinitrobenzene), Debsyl or Densyl chloride reagents are used to primarily
identify the amino-terminal residue. By using this method one can find the number of
subunits having free amino terminal end.
b. The Edman degradation procedure labels and removes only the amino-terminal
residue from a peptide, leaving all other peptide bonds intact.
The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions,
which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC)
adduct. The peptide bond next to the PTC adduct is then cleaved in presence of
trifluoroacetic acid, with removal of the amino-terminal amino acid as an
anilinothiazolinone derivative. The derivatized amino acid is extracted with organic
solvents, converted to the more stable phenylthiohydantoin derivative by treatment
with aqueous acid, and then identified.
The sequential reactions are carried out under first basic and then acidic conditions to
control the entire process. Each reaction with the amino-terminal amino acid can go
essentially to completion without affecting any of the other peptide bonds in the
peptide. The process is repeated until, typically, as many as 40 sequential amino acid
residues are identified. The reactions of the Edman degradation have been automated.
Breaking of disulfide bonds in proteins
Two common methods are illustrated.
Oxidation of a cystine residue with
performic acid produces two cysteic
acid residues.
Reduction by dithiothreitol (or β-
mercaptoethanol) to form Cys residues
must be followed by further
modification of the reactive -SH groups
to prevent re-formation of the disulfide
bond. Carboxymethylation by
iodoacetate serves this purpose.
Mass Spectrometry an Alternative Method to Determine Amino Acid
Sequences
When the newly charged molecules are introduced into an electric and/or magnetic field, their paths depends on their mass-
to-charge ratio, m/z. This property of the ionized species can be used to deduce the mass (m) with very high precision
The proteins placed on a light absorbing matrix are ionized with a short pulse of laser light and then desorbed from the
matrix into the vacuum system. This process, known as matrix-assisted laser desorption/ionization mass spectrometry, or
MALDI MS, has been successfully used to measure the mass of a wide range of macromolecules.

In a second and equally successful method, macromolecules in solution are forced directly from the liquid to the gas phase.
A solution of analytes is passed through a charged needle that is kept at a high electrical potential, dispersing the solution
into a fine mist of charged microdroplets. The solvent surrounding the macromolecules rapidly evaporates, leaving multiply
charged macromolecular ions in the gas phase. This technique is called electrospray ionization mass spectrometry, or ESI
MS. The m/z of the molecule can be analyzed in the vacuum chamber.
Applications: This techniques require very less amounts of sample, so they can be readily applied to the small amounts of protein that can be
extracted from a two-dimensional electrophoretic gel. More accurate molecular mass of a protein can be measured, it is convenient and
accurate method for detecting changes in mass due to the presence of bound cofactors, bound metal ions, covalent modifications, and so on.
The Structure of Proteins

Primary structure is the sequence of amino acid

residues. All covalent bonds (mainly peptide bonds
and disulfide bonds) linking amino acid residues in
a polypeptide chain is its primary structure.

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.

When a protein has two or more polypeptide

subunits, their arrangement in space is referred to
as quaternary structure
 Introduction about protein structure
• The spatial arrangement of atoms in a protein or any part of a protein is called its conformation.
• A change in protein conformation could occur by rotation about single bonds without breaking it.
• Of the many conformations that are theoretically possible in a protein containing hundreds of single bonds, only a few
generally predominate under biological conditions.
• Why there are multiple stable conformations of proteins? Because most proteins bind to other molecules or catalyze
reactions.
• A Protein’s Conformation Is Stabilized Largely by Weak Interactions.
• Many proteins do not have disulfide bonds because the environment within most cells is highly reducing due to high
concentrations of reductants such as glutathione, and most sulfhydryl groups will thus remain in the reduced state.
• Outside the cell, the environment is often more oxidizing, and disulfide formation is more likely to occur.
• In eukaryotes, disulfide bonds are found primarily in extracellular proteins.
• Disulfide bonds are also uncommon in bacterial proteins. However, thermophilic bacteria, as well as the archaea, typically
have many proteins with disulfide bonds to stabilize proteins. This is presumably an adaptation to life at high temperatures.
For all proteins of all organisms, weak interactions are especially important in the folding of polypeptide chains into
their secondary and tertiary structures. The association of multiple polypeptides to form quaternary structures also
relies on these weak interactions.
 Peptide bond is rigid and planar
• In the late 1930s, Linus Pauling and Robert Corey performed a series of studies
to understand the protein structure. They began with a careful analysis of the
peptide bond.
• The α carbons of adjacent amino acid residues are separated by three covalent
bonds, arranged as Cα—C—N—Cα
• X-ray diffraction studies showed that the peptide C—N bond is somewhat
shorter than the C—N bond in a simple amine.
• This indicated partial sharing of two pairs of electrons between the carbonyl
oxygen and the amide nitrogen, setting up a small electric dipole.
• The six atoms of the peptide group lie in a single plane.
• From these findings Pauling and Corey concluded that the peptide C—N bonds,
because of their partial double-bond character, cannot rotate freely. Rotation is
permitted about the N—Cα and the Cα—C bonds. The backbone of a
polypeptide chain can thus be pictured as a series of rigid planes, with
consecutive planes sharing a common point of rotation at Cα. The rigid peptide
bonds limit the range of conformations possible for a polypeptide chain.
• In principle, phi and psi can have any value between -
180º and +180 but many values are prohibited by
steric interference between atoms in the polypeptide
backbone and amino acid side chains.
• The conformation in which both phi and psi are 0º is
prohibited for this reason; this conformation is used
merely as a reference point for describing the angles of
rotation. Allowed values for phi and psi are graphically
revealed when psi is plotted versus phi in a
Ramachandran plot (as shown in figure) introduced by
G. N. Ramachandran.
Some examples of protein secondary structures
 The alpha-Helix Is a Common Protein Secondary Structure
(The angstrom, Å, named after the physicist Anders J. Ångström, is equal to 0.1
nm.

The simplest arrangement the polypeptide chain could assume with its rigid
peptide bonds (but other single bonds free to rotate) is a helical structure, which
Pauling and Corey called the alpha-helix.

In this structure the polypeptide backbone is tightly wound around an imaginary

axis drawn longitudinally through the middle of the helix, and the R groups of the
amino acid residues protrude outward from the helical backbone.

The repeating unit is a single turn of the helix, which extends about 5.4 Å along
the long axis. The amino acid residues in an alpha-helix have conformations with
psi = - 45º to - 50º and phi = - 60º, and each helical turn includes 3.6 amino acid
residues.

The alpha helix is predominant structure in alpha-keratins. More generally, about

one-fourth of all amino acid residues in polypeptides are found in alpha helices,
the exact fraction varying greatly from one protein to the next.

The structure is stabilized by a hydrogen bond between the hydrogen atom

attached nitrogen atom of a peptide linkage and the carbonyl oxygen atom of the
fourth amino acid on the amino-terminal side of that peptide bond.
Some examples of protein secondary structures
 The alpha-Helix Is a Common Protein Secondary Structure
The simplest arrangement the polypeptide chain could assume with its rigid
peptide bonds (but other single bonds free to rotate) is a helical structure, which
Pauling and Corey called the alpha-helix.

In this structure the polypeptide backbone is tightly wound around an imaginary

axis drawn longitudinally through the middle of the helix, and the R groups of
the amino acid residues protrude outward from the helical backbone.

The repeating unit is a single turn of the helix, which extends about 5.4 Å along
the long axis. The amino acid residues in an alpha-helix have conformations
with psi = - 45º to - 50º and phi = - 60º, and each helical turn includes 3.6
amino acid residues.

The alpha helix is predominant structure in alpha-keratins. More generally,

about one-fourth of all amino acid residues in polypeptides are found in alpha
helices, the exact fraction varying greatly from one protein to the next.

The structure is stabilized by a hydrogen bond between the hydrogen atom

attached nitrogen atom of a peptide linkage and the carbonyl oxygen atom of
the fourth amino acid on the amino-terminal side of that peptide bond.

(The angstrom, Å, named after the physicist Anders J. Ångström, is equal to 0.1 nm)
Effect of Pro or Gly residues on the structure of alpha helix

In proline, the nitrogen atom is part of a rigid ring, and

rotation about the N-C alpha bond is not possible.
Thus, a Pro residue introduces a destabilizing kink in an
alpha helix.
In addition, the nitrogen atom of a Pro residue in peptide
linkage has no substituent hydrogen to participate in
hydrogen bonds with other residues. For these reasons,
proline is only rarely found within an alpha helix.

Glycine occurs infrequently in alpha helices for a different

reason: it has more conformational flexibility than the other
amino acid residues.
Some Amino Acid residues Affects the stability of alpha-Helix

Not all polypeptides can form a stable alpha-helix. Interactions

between amino acid side chains can stabilize or destabilize this
structure. For example, if a polypeptide chain has a long block of
Glu residues, this segment of the chain will not form an alpha-helix
at pH 7.0. The negatively charged carboxyl groups of adjacent Glu
residues repel each other so strongly that they prevent formation of
the alpha-helix. For the same reason, if there are many adjacent
Lys and/or Arg residues, which have positively charged R groups
at pH 7.0, they will also repel each other and prevent formation of
the alpha-helix. The bulk and shape of Asn, Ser, Thr, and Cys
residues can also destabilize an alpha-helix if they are close
together in the chain.
Five different kinds of factors affect the stability of an alpha-helix:

(1) the electrostatic repulsion (or attraction) between successive

amino acid residues with charged R groups,
(2) the bulkiness of adjacent R groups,
(3) the interactions between R groups spaced three (or four)
residues apart,
(4) the occurrence of Pro and Gly residues, and
(5) the interaction between amino acid residues at the ends of the
helical segment and the electric dipole inherent to the alpha-
helix. The tendency of a given segment of a polypeptide chain
to fold up as an alpha-helix therefore depends on the identity
and sequence of amino acid residues within the segment.
 Exercise
What is the length of a polypeptide with 80 amino
acid residues in a single, continuous α helix?
Solution:
Solutionm: An idealized α helix has 3.6 residues per
turn, and the rise along the helical axis is 5.4 Å. Thus,
the rise along the axis for each amino acid residue is
1.5 Å. The length of the polypeptide is therefore 80
residues × 1.5 Å/residue = 120 Å.
Beta-conformation or sheet is another type of repetitive structure
found in proteins, predicted by Pauling and Corey
This is a more extended zigzag conformation of polypeptide chains,
rather than helical structure.
The zigzag polypeptide chains can be arranged side by side to form a
structure resembling a series of pleats.
In this arrangement, called a beta sheet, hydrogen bonds are formed
between adjacent segments of polypeptide chain. The individual segments
that form a beta sheet are usually nearby on the polypeptide chain.
The R groups of adjacent amino acids protrude from the zigzag structure
in opposite directions.
The adjacent polypeptide chains in a beta sheet can be either parallel or
antiparallel (having the same or opposite amino-to-carboxyl orientations,
respectively).
Beta-Keratins such as silk fibroin and the fibroin of spider webs have a
very high content of Gly and Ala residues, the two amino acids with the
smallest R groups.
 Beta Turns Are Common in Proteins
In globular proteins, which have a compact folded structure,
nearly one-third of the amino acid residues are in turns or loops
where the polypeptide chain reverses direction. These are the
connecting elements that link successive runs of helix or beta
conformation. beta turns are more common at the ends of
antiparallel Beta sheet.
The structure is a 180º turn involving four amino acid residues,
with the carbonyl oxygen of the first residue forming a hydrogen
bond with the amino-group hydrogen of the fourth. The peptide
groups of the central two residues do not participate in any
interresidue hydrogen bonding.
Gly and Pro residues often occur in beta turns, because Gly is
Structures of beta turns.
small and flexible and imino nitrogen of proline readily assume
(a) type I turns occur more than twice as frequently as type II.
the cis configuration, to form tight turn. Of the several types of
(b) Type II turns always have Gly as the third residue.
beta turns, the two shown are the most common. The hydrogen bond between the peptide groups of the first and
fourth residues of the bends.