INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

Biochemistry

Unit 2-III
Protein: Structure, Function and Analysis
Purification and analysis of proteins
• In order to study biochemical and biophysical properties, a protein must be
purified. Proteins can be purified using many methods including
chromatographic techniques. Proteins can be purified on the basis of charge,
size and affinity by ion-exchange, gel filtration and affinity chromatography
techniques respectively.
• SDS-PAGE can be used for analyzing the purity of the protein and for
determination of molecular mass. Centrifugation techniques are used to
analyze and purify proteins that differ in mass or density. Mass spectrometry is
a very powerful technique to analyze proteins and peptides. It is routinely used
to measure mass of proteins.
• The activity of the protein could be determined using standard assays or
developing an assay for protein activity. The spectroscopy and other techniques
are employed for the same.
• Secondary structure determination can be performed by circular dichroism
studies. It is also used to analyze the stability of protein in different conditions.
X-ray crystallography, NMR and Cryo-electron microscopy are used for
determining three-dimensional structure of proteins.
2
Purification and analysis of proteins
• The source of the protein to be purified and characterized could be plants,
animals, microbes etc.
• Let’s assume, a protein from a bacterial source (may be after cloning and
expression in a particular bacterial host like E.coli) is to be purified and
characterized. The first step will be to lyse the cell and prepare a crude extract.
The bacterial cells will be collected after centrifugation and then re-suspended
in appropriate buffer for lysis. The different methods of lysis could include
Sonication, French Press, Enzyme+detergent, crude methods like repeated
Freeze and Thaw cycle and others. The soluble proteins will be in supernatant
and the cell debris will be discarded after centrifugation or dialysis. The clear
solution containing impure protein will be taken for purification and
characterization

• Various methods employed for purification and analysis will include,

Electrophoresis; Column Chromatography techniques; Protein sequencing;
Centrifugation; Mass Spectrometry; Circular dichroism; X-ray Crystallography
NMR and Cryo-electron microscopy.

3
 Centrifugation
• Centrifugation is one of the most important and widely
applied research techniques in biochemistry, cellular and
molecular biology. Current research and clinical
applications rely on isolation of cells, sub cellular
organelles, and macromolecules, often in high yields.

• This separation technique exploits the inherent varied

sedimentation property of substances for their isolation by
the application of centrifugation field. It is used to
separate or concentrate materials suspended in a liquid
medium. The resulting solution has two components
namely the Sediment (Pellet), and Supernatant.
The cells are disrupted and
• The theoretical basis of this technique is the effect of homogenate is fractionated by
centrifugation yielding pellet at the
gravity on particles (including macromolecules) in
bottom of tube and supernatant.
suspension. Two particles of different mass, shape or The clear supernatant can be
density will settle in a tube at different rates in response to obtained by again centrifuging
gravity. Centrifugal force is used to increase this settling supernatant at higher centrifugal
force and then can be used for
rate in an instrument called a centrifuge. purification of soluble protein of
interest.

4
Centrifugation
• A centrifuge uses centrifugal force (g-force) to isolate suspended particles from
their surrounding medium on either a batch or a continuous-flow basis. Rate of
sedimentation depends on this applied centrifugal field, G, being directed
radially outwards and is determined by G = ω2r.
• The rate of movement of a particle can be expressed as sedimentation coefficient,
s, [s = m(1- ῡρ)/f, where m is mass of the particle; ῡ is partial specific volume (reciprocal of particle
density); ρ is the density of medium; and f is the frictional coefficient]. Sedimentation coefficient
is usually expressed in Svedberg units (S) equal to 10-13s. The smaller the S value,
slower is the movement under a centrifugal field.
• The sedimentation velocity of a particle depends in part on mass and density.
Shape, also, influences the sedimentation velocity as it affects viscous drag.
Elongated particle sediment slower than spherical ones of same mass.
• The sedimentation velocity also depends on the density of the solution. Particles
will sink when ῡρ < 1, will float when ῡρ > 1and will not move when ῡρ = 1.
• The various types of centrifugation techniques include differential and density
gradient centrifugation. Ultracentrifugation can be used for separating
biomolecules and analyzing their mass, density and shape.

5
Column Chromatography
• A separation technique in which
stationary phase/stationary phase on a
suitable matrix is packed in a column
made of glass, metal or polypropylene.
The mobile phase passes through the
column either by gravity feed or by use
of pumping system. This method has
become the most commonly used
mode of chromatography.

• In column chromatography, the

components in a mixture are separated
on the basis of their different
distribution coefficient in two phases
(stationary and mobile phase). A
mixture of two compounds with
considerably different distribution
coefficients would separate rapidly into
distinct bands in the column.

6
Gel-filtration/size-exclusion chromatography
• Separation is based on protein size. Dextran or polyacrylamide beads of
uniform diameter are manufactured with different pore sizes. Depending on
the sizes of the proteins to be separated, they will enter the pore if small
enough, or be excluded if they are too large.
• The applications of gel-filtration chromatography includes purification, relative
molecular mass determination, solute concentration, desalting and protein-
protein or protein-ligand binding studies.
Small molecules can
enter the beads and
spent more time in
column and eluted last
as compared to large
molecules which can
not enter the beads
and are eluted in void
volume. The molecules
with intermediate size
are eluted at an
intermediate positions

7
Ion-exchange Chromatography
• Separation of proteins based on the net charge (Cation
and Anion Exchange Chromatography) of their
constituent amino acids at a particular pH. Different salt
concentrations/pH gradient can be used to elute the
bound proteins into tubes in a fraction collector. The
resins for binding (+) or (-) charged proteins can be used.

Cation-exchanger Anion-exchanger
with exchangeable with exchangeable
counter ions. (+) charge
counter ions.
proteins

(-) charge
proteins

8
Affinity Chromatography
• Affinity chromatography separates proteins on
the basis of a reversible interaction between a Protein recognized
protein (or group of proteins) and a specific by antibody
ligand coupled to a chromatographic matrix.
Protein not
Based on the target proteins ability to bind a
recognized by
specific ligand, only proteins that bind to this antibody
ligand will be retained on the column bead. This
is especially useful for immunoaffinity
purification of proteins using specific antibodies
for them.
• With high selectivity, hence high resolution, and
high capacity for the protein(s) of interest,
purification levels in the order of several
thousand-fold with high recovery of active
material are achievable. Target protein(s) is
collected in a purified, concentrated form.
• Some of the typical biological interactions frequently used in affinity chromatography
include, Enzyme-inhibitor/substrate analogue; Antibody-antigen; Lectin-
glycoprotein/polysaccharides; Hormone-receptor; Metal ions-poly(His) fusion
proteins etc.
9
SDS-PAGE
• Electrophoresis method is based on the movement of charged particles under
the influence of an electric field. The biological molecules can be separated
using electrophoresis as they possess ionizable groups and can exist either as
cations or anions at a particular pH and migrate to cathode or anode.
• In gel electrophoresis, proteins are generally analyzed on acrylamide gels
(copolymerization of acrylamide and bis-acrylamide in presence of TEMED and
APS) under native or denaturing conditions. The SDS-PAGE (sodium dodecyl
sulfate-polyacrylamide gel electrophoresis), a discontinuous system developed
by Laemmli, is the method of choice in Biochemistry Labs for routine protein
analysis.
• Presence of SDS, a detergent, denatures and linearizes a protein (Na and
sulfate bind to charged amino acids, the hydrocarbon chain interacts with
hydrophobic residues). An applied electric field leads to separation of proteins
based on size through a defined gel pore matrix.
• For electrophoresis in the absence of SDS, separation is based on size, charge
and shape of the protein (proteins are not denatured and can potentially retain
function or activity).
10
SDS-PAGE
• Separation of proteins based on their size is linear in relation to
the distance migrated in the gel. Using protein standards of
known mass and staining of the separated proteins with dye,
the mass of the proteins in the sample can be determined. This
is useful for purification and diagnostic purposes.
• The SDS-PAGE is performed after every purification step to
assess the level of purification till a single band is observed, an
indication of a very high level of purity.

11
Primary structure
• Protein sequencing strategy:
1. Separation of polypeptide chains

2. Cleavage of disulfide bridges: O

a) Oxidative cleavage by reaction with performic acid (H-C-O-OH )
S S Performic acid SO3- SO3- (Cysteic acid residues)
b) Reductive cleavage with sulphydryl compounds such as 2-mercaptoethanol or
DTT. Then modification of –SH group by alkylation with iodoactate or modification
with 3-bromopropyl amine.
S HSCH2CH2OH SH + S-CH2-CH2-OH
S 2 mercaptoethanol SH S-CH2-CH2-OH
SH
SH + ICH2COOH HI + S-CH2-COO-
SH Iodoacetic acid S-carboxymethyl erivative
SH + BrCH2CH2CH2NH2 HBr + S-CH2CH2CH2NH
3 3-Bromopropyl amine

12
Primary structure
3. Analysis of amino acid composition:
Peptide 6N HCl Amino acids Ion-exchange Sulfonated Elution
110 °C, 24 h Column polystyrene volume

+ Ninhydrin (quantitation) Blue colour

Ninhydrin can detect micrograms (10 nmol). Fluorescamine (in place of ninhydrin) can
detect nanograms (10 pmol) which reacts with a-amino group to give highly
fluorescent product.
AA1-AA2-AA3-AA4-Peptide
4. Identification of N- and C-terminal residues:
Labelling
Edman degradation (developed by Pehr AA1-AA2-AA3-AA4-Peptide
Edman): a method of sequencing amino acids in a Release
peptide where the amino-terminal residue is AA1 + AA2-AA3-AA4-Peptide
labeled and cleaved from the peptide without Labelling
disrupting the peptide bonds between other amino AA2-AA3-AA4-Peptide
acid residues. Release
AA2 + AA3-AA4-Peptide

13
Primary structure
Edman degradation
H O O-
N=C=S + H2N-C-C-Gly-Asp-Phe-Arg-Gly-C=O
CH3
Phenyl isothiocynate Labelling

S HH O O-
N=C N-C-C-Gly-Asp-Phe-Arg-Gly-C=O
CH3
Release
N C S
O-
C N H H2N-Gly-Asp-Phe-Arg-Gly-C=O
O (Peptide shortened by one residue)
C
H CH3
PTH-alanine
(Phenylthiohydantoin)

14
Primary structure
C-terminal analysis:
Carboxypeptidase cleave amino acid residues from the C-terminal end of polypeptide chain

- Carboxypeptidase A (bovine pancreas) - All residues except Pro, Arg and Lys
- Carboxypeptidase B (Hog pancreas) - Only when Arg and Lys at C-terminal
- Carboxypeptidase C (Citrus leaves) - Any C-terminal residue
- Carboxypeptidase Y (Yeast) - Any residue

5. Fragmentation of the polypeptide chain:

Chemical cleavage Enzymatic cleavage
Cyanogen Bromide Carboxyl side of Met Trypsin Carboxyl side of Arg & Lys
O-Iodosobenzoate Carboxyl side of Trp Clostripain Carboxyl side of Arg
Hydroxylamine Asn-Gly bond Thrombin Carboxyl side of Arg
2-Nitro-5-thiocyanato Chymotrypsin Carboxyl side of large
-benzoic acid Amino side of Cys hydrophobic side chain

Carboxypeptidase A Amino side of C-terminal

residue except Pro Arg Lys
15
Primary structure
6. Reconstruction of overall amino acid sequence:
• Edman degradation can determine around 50 residues in a protein and therefore,
peptide obtained by specific cleavage can not be longer than about 50 residues.
• The peptides obtained by specific chemical or enzymatic cleavage can be
separated by chromatography and then sequenced. The complete sequence of a
protein can be determined by overlap peptides.

Protein A (Ala2, Gly, Asp, Met2, Thr, Leu, Lys2, Val, Asn, Glu, Ser)
Trypsin Digestion & Cyanogen Bromide
Edman degradation
Val-Asn-Ala-Glu-Met-Ser-Lys Ala-Gly-Asp-Met
Ala-Gly-Asp-Met-Thr-Leu-Lys Thr-Leu-Lys-Val-Asn-Ala-Glu-Met
Arrangement of
fragments

Ala-Gly-Asp-Met -Thr-Leu-Lys-Val-Asn-Ala-Glu-Met-Ser-Lys-Ala
The peptides obtained by cyanogen bromide digestion overlaps peptides obtained from tryptic digestion

7. Location of disulfide cross bridges:

16
 Mass Spectrometry
• Mass spectrometry can be used for determining of mass and sequence of a protein.
It is used for identification of peptides and proteins

• Basic operation of the technique:

- Evaporate and ionize molecules in vacuum creating gas phase ions.
- Separate ions in space and (or) time based on their m/z ratio.
- Measure the amount of ions with specific m/z ratio.
- Macromolecular ionization methods in Mass Spectrometry include Electrospray ionization (ESI-
MS), Fast-Atom bombardment (FAB-MS), Laser ionization (LI-MS), Matrix assisted laser
desorption/ionization – Time of flight (MALDI-TOF)

17
Secondary structure
•Circular Dichroism (CD) method is based on the differential absorption of left and right circularly
polarized light by optically active chiral molecules. Many biological compounds are optically
active. Application to biological molecules include determination of protein’s secondary
structure, determination of optical purity, analysis of the conformational changes in a protein,
comparison of the structure of wild type and mutant proteins, nucleic acid structure and
changes upon binding or melting. The Far-UV (195-250 nm) CD spectrum of proteins can reveal
important characteristics of their secondary structure. In ‘Near-UV’ (250-350 nm) CD spectrum,
chromophores are aromatic amino acids and disulfide bonds (250-270 nm, phenylalanine; 270-
290 nm, tyrosine; 280-300 nm, tryptophan).
10

MRE X 103 (deg cm2 dmol-1 )

5

0
VII
VI
-5

-10
V
-15
IV
-20
I
-25
200 205 210 215 220 225 230 235 240
Wavelength nm

Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984

18
 Tertiary and quaternary structure
• The tertiary and quaternary structure of a protein can be determined by X-ray crystallography,
NMR and Cryo-electron microscopy.
• In X-ray crystallography, a purified protein is crystalized to obtain X-ray diffraction and
subsequently tertiary/quaternary structure.

19
X-ray crystallography
• A method for determining the arrangement
of atoms within a crystal.
• A beam of X-rays strikes a crystal and gets
diffracted.
• From the angles and intensities of these
diffracted beams, a crystallographer can
produce a map of the electron density
within the crystal.
• From the electron density map, the mean
positions of the atoms in the crystal can be
determined. Crystals are made of infinite
number of unit cells. Unit cell is the smallest
unit of a crystal, which, if repeated, could
generate the whole crystal.
• The unit cell dimensions are defined by six
numbers, the lengths of the three axes, a, b,
and c, and the three inter axial angles a, 
and .
20
Nuclear Magnetic Resonance (NMR)
• Nuclear Magnetic Resonance (NMR)
spectroscopy is one of the most powerful tool
available for determining the structure of
organic compounds including biological
macromolecules.
• The technique relies on the ability of atomic
nuclei to behave like a small magnet and
align themselves with an external magnetic
field. The nuclei from isotopes of many
• When irradiated with a radio frequency signal elements (1H,13C, 31P, 15N, 19F etc) possess
the nuclei in a molecule can change from an overall spin.
being aligned with the magnetic field to
being opposed to it.
• It is called “nuclear” as the instrument works
on stimulating the “nuclei” of the atoms to
absorb radio waves. The energy frequency at
which this occurs can be measured and is
displayed as an NMR spectrum.

21
Nuclear Magnetic Resonance (NMR)
Chemical Shift and the TMS Standard
• Chemically different protons have different
electronic environments.
• Differences in the electronic environments
cause the protons to experience slightly
different applied magnetic fields owing to the
shielding/deshielding effect of the induced
electronic magnetic fields.
• The standard reference is tetramethylsilane
(TMS). The compound has four methyl groups
bonded to a silicon. This association with the
reference signal is called the chemical shift and
is measured in parts per million (ppm).
• After the samples have been referenced to
the TMS resonance at zero ppm, the actual
NMR peak position in Hz is divided by the
resonance frequency of the spectrometer,
which is in MHz.
22
23