Methods of protein structure determination

1. By :-
1. Assistant Professor
Mr. Rajesh Kumar Kushwaha
Ph.D(Biochemistry), M.Tech(BT), M.Phil(BT),
M.Sc(Biochemistry), B.Ed(Science) , D.Pharm,
GATE,CSIR NET
Dept. Of Biotechnology Engineering
Summaryof methodsfor proteinstructure
anddetermination

2. ProteinMajor

3. Proteins were discovered by J.J
Berzelius in 1838 .The word
"Protein" is derived from a Greek
word "protas" meaning "of
primary importance," because of
the fundamental role of proteins
in sustaining life.

4. Protein is a substance that has amino
acids as basic building block
compounds and carbon, hydrogen,
oxygen, nitrogen and sometimes
sulfur and is found in many foods. An
example of a protein is the type of
nutrient found in meats.

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General
Structure
Amino acids are molecules
used to build proteins due to
which the Amino acid are called
the building block of protein.
All amino acids have a central carbon atom surrounded by a hydrogen atom,
a carboxyl group (COOH), an amino group (NH2), and an R-group. It is the R-
group or side chain that differs between the 20 amino acids.

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Types of Amino Acid
Non-Essential Amino Acids
Non-Essential amino acids can
be made by the body
e.g.
Essential Amino Acids
Essential amino acids cannot be
made by the body so you must get
them from your diet.
e.g.
8. Isoleucine Methionine Arginine, Valine
,Histidine, Isoleucine ,Leucine Lysine
,Phenylalanine ,Threonine Tryptophan
Alanine Arginine
Glutamic Glycine serine,
cysteine, aspartate,
asparagine, glutamate,
glutamine, tyrosine and
proline.

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SuAceid
7 Major Classes of Proteins
• Structural e.g. Hair.
• Contractile e.g. Actin(muscle cells)
• Storage e.g. Egg whites.
• Defense e.g. Antibodies.
• Transport e.g. Hemoglobin.
• Signaling e.g. Hormones
• Enzymes e.g. Lactase

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Hi• Poultry and fish
• Eggs
• Dairy products like milk, yoghurt and cheese
• Seeds and nuts
• Beans and legumes (such as lentils and chickpeas)
Some sources of dietary Protein
include:

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PPrreooitne
in
Structu
re
Tpnes
of
Typical bonds in protein molecules
Peptide bond
Covalent bonds
Disulfide bond
Hydrogen bond
Non-covalent bonds
Ionic bond
Hydrophobic
interactions

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Peptide Bonds (covalent in nature)
• Amino Acids Are Linked by Peptide Bonds to Form
Polypeptide Chains.
• Proteins are linear polymers formed by linking the α- carboxyl
group of one amino acid to the α-amino group of another amino
acid with a peptide bond (also called an amide bond).

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Hydrogen bonds
• The hydrogen-bond also play a very important roles
in proteins' structure because it stabilizes the
secondary, tertiary and quaternary structure of
proteins which formed by alpha helix, beta sheets,
turns and loops.
• The hydrogen-bond connected the amino acids
between different polypeptide chains in proteins
structure.

12. HistorydefinitionSourcesDifferent

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oni c
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Differe
t
y
PPrreooitne
in
Structu
re
Tpnes
of Ionic Bonds
Electrostatic interactions occur
between two oppositely charged
molecules.
Disulfide Bridges
A disulfide bond can be form between
two cysteines( C3H7NO2S) through
oxidation. These are also the strongest
covalent bonds within a protein's tertiary
structure.
Hydrophobic Interaction
• The hydrophobic interaction
originates from the tendency
of non-polar molecules to
minimize their interactions
with water.
• When non-polar molecules
interact with water, these
molecules tend to cluster
together in the center to form
a micelle.

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Prote
in
Structural Organization
Primary
Structure
•Secondary
Structure
•Tertiary
Structure
•Quarterly
Structure

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Prote
in
Funct
ioTpnes
of
Primary structure
• Describe the number and sequence of
Amino acid in the protein molecule.
• F.Sanger first describe the sequence and
the number of the amino acids in the
protein molecule.
• He concluded that
• Insulin is made up of 51 amino acid in two
chain.
• These two chains are held together by
Disulphide Bond
• Hemoglobin is made up of four chains two
alpha and two beta.
• Alpha chains contain 141 amino acid and
each beta chains contains 146.

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The size of the protein molecule is determine
by the type of the amino acid and the number
of the amino acids present in the structure of
the protein.

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Prote
in
Secondary
structure
• Within the long protein chains there are regions in
which the chains are organized into regular
structures known as alpha-helixes ,beta-
pleated sheets.
• These are the secondary structures in proteins.
• It forms the spiral formation of the basic
polypeptide chain
• The helix is uniform throughout the structure
containing 3.6 amino acid in each turn.
• These secondary structures are held together by
hydrogen bonds.
• The beta plated sheet is formed by the folding

18. HistorydefinitionDifferent
FunctioTypnesof
Protein

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Prote
in
Funct
ioTpnes
of
Tertiary
structure
• Polypeptide chain bends and folds upon its- self
forming globular protein.
• Structure is maintained by the different types of
the bonds
• Ionic, covalent, and disulphide bond.
• The three-dimensional structure of a protein or
nucleic acid.
• Amino acids form secondary structures such as
alpha helices, beta sheets, and random coils,
which in turn fold on themselves to form the tertiary
structure of the protein.

20. definitionSourcesProtein Structure
Typesof Protein

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Prote
in
Funct
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o
Quaternary
structure
•Proteins have primary, secondary, tertiary,
and quaternary structure.
•Quaternary structure is the only one, which
involves multiple protein subunits
•A variety of bonding interactions including
hydrogen bonding, salt bridges, and
disulfide bonds hold the various chains into
a particular geometry.

22. HistorydefinitionDiffrent
Typnesof
Protein

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Fibrous protein
• Contain one or more
polypeptide in the form
of fibrils
• Insoluble in water
• Secondary structure is
the main component
• They are elastic in nature
• Play vital role in the
maintenance of structure
of the cell and cell
organelles
Exampl
e• Silk fiber (from the
silk warm and
spider web)
Myosin (in muscle
cells)
• Fibrin (of blood cells)
• Keratin (nail and
hair)

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Globular protein
• These are spherical or elliptical in structure this
is because of the multiple folding of the
polypeptide chain
• A variety of bonding interactions including
hydrogen bonding, salt bridges, and disulfide
bonds hold the various chains into a particular
geometry.
• Tertiary structure is most important Globular
proteins are somewhat soluble in the aqueous
solution of acids, salt and aqueous alcohol
• They change their structure when structure or
physical or physiological changes takes place
• Enzyme
• Antibodie
s
• Hormone
s

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Important Functions of Protein
in Your Body
•Growth and Maintenance. Your body needs
protein for growth and maintenance of
tissues.
•Causes Biochemical Reactions.
•Acts as a Messenger.
•Provides Structure.
•Maintains Proper pH.
•Balances Fluids. (Across the membrane)
•Bolsters Immune Health.
•Transports and Stores Nutrients.

26. Outline
Introduction
X-ray crystallography
Nuclear magnetic
resonance Cryo-Electron
microscopy

27. Introduction
Protien structure determination:
Determining 3D structure of protein help us to
understand mechanism of action of protein
and it’s functions.

28. Protein X-ray crystallography
o Molecule size is too small to see under light microscope as
it’s wave length(400-700nm)is much larger than molecule size.
o we use x-ray radiation to look to molecules as it’s
wave length(0.1nm) is much smaller.
 X-ray crystallography principle:
-That X-rays are diffracted by crystal.
-Direct detection of atom position
in crystal .

29. Protein X-ray crystallography
o Steps:
1.Protein purification:
Minimum 5 to 10 mg pure soluble protein are
required with better than 95% purity.
2.Protein crystallization:
1. Disorder of unit cell.
2. Vibration of molecule.
3. Distortion in crystallization.

30. Protein X-ray crystallography
Crystallization –Hanging Drop Method:
1 to 5μl protein solution is suspended over 1 ml
reservoir containing precipitant solution.
3.Date collection:
-Mounting crystal…
-Crystals mounting by rotating them and X-ray
beams passed through them.
-This method include using a capillary or tube.
-Both capillary and tube mounted in goniometer.

31. o The X-ray source is often synchrotron(has high resolution).
o The typical size of crystal is(0.3x0.3x0.1nm).
o X-rayes penetrate crystal ,scattered and captured as diffraction pattern on a detector.
o Rotate crystal and repeat the process in different degree until
(30degree or180degree)and record XRDs pattern which Used to form
electron density map of crystal.
o This produced electron map may has some phasing problem can be treated by:
1. Molecular replacement.
2. Isomorphous replacement.
3. Fourier transform.
 Fitting protein sequence to get better electron map.

32. Nuclear magnetic resonance(NMR)
o The aim:Measure set of distance between atomic nuclei.
• Used for protein that are hard to crystallize or can be
dissolved at high concentration.
• NMR principle:
-Based on nucleus spin( have angular momentum vector).
-Spin can be parallel,anti parallel external magnetic
field(forms energy state(low, high)).
-Applying radiofrequency change this state.

33. Nuclear magnetic resonance(NMR)
o Steps:
1.Protein solution:
Highly purified protein solution(300-600µl with protein conc.(0.1-3ml.M).
2.Data collection:
Distinct nucleus produce chemical shift in two main experiments category:
-One where magnetization is transferred through the chemical bonds.
-One where the transfer is through space.
3.Sequential resonance assignment:
- Map chemical shift to atom by sequential walking.
- Take the advantage of the known protein sequence.
- The assignment based on proton/proton NOEs
observed in is quite time consuming.
http://chemistry.stackexchange.com/questions/42757/why-only-one-
peak-is-observed-in-nmr-spectrum-of-h2

34. Nuclear magnetic resonance(NMR)
4.Collection of conformational constraints:
Geometric conformational information derived fromNMR..
1.Distance between nuclei.
2.Angles between bonds.
3.Motion in solution.
Chemical shift date provide information on the type of 2ry structure.
5.Structure calculation:
-Determined restraints is the input which used by computer programs
-This process give us ensemble of structure.

35. Cryo-Electron microscopy
o It is a new technology for studying the
architecture of cells, viruses and protein
assemblies at molecular resolution.
• Principle of Cryo-EM
When a beam of electrons is passed through
specimen a part of it is transmitted and this
part when projected on fluorescent
screen its image can be seen by the
observer.
o Biological specimen:
1.Thin film.
2.Vitreous section.

36. Cryo-Electron microscopy
Advantages Disadvantages
1. Allows the observation
of specimens that
have not been
stained or fixed in any
way.
2. Showing them in
their native
environment.
3. Less in
functionally
irrelevant
1. Expensive.
2. The resolution of
cryo- electron
microscopy maps
is not high
enough.

37. Thanks
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