Protein stability

Outline
1Introduction and evolution of protein
2 Level of protein
• Primary secondary tertiary and quaternary protein.
3 Stability and protein folding
4 Function of protein
• Storage, transport, defensive system. cell signaling
5 Protein purification
6 Protein determination method
• X ray crystallography
• NMR

Protein Introduction and
evolution

Introduction
Proteins are linear polymers built of monomer
units called amino acids.
Proteins contain a wide range of functional
groups.
functional groups include alcohols, thiols,
thioethers, carboxylic acids, carboxamides, and a
variety of basic group

Essential amino acid
Essential amino acids cannot be made by the body. As a result, they
must come from food.
The 9 essential amino acids are: histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
Non essential amino acid
amino acid that can be made by humans and so is not essential to the
human diet. There are 11 nonessential amino acids:
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, proline, serine, and tyrosine.

Peptide bond
 Two or more amino acids are joined by condensation reaction
i.e, removal of water molecule forms peptide bond
 The partial double-bond character of the peptide bond defines
the conformations a polypeptide chain may assume.
 It is shorter then a single bond
 Rigid & planar.

Introduction
Proteins can interact with one another and with other
biological macromolecules to form complex
assemblies
assemblies include macro-molecular machines that
carry out the accurate replication of DNA, the
transmission of signals within cells, and many other
essential processes.

Evolution
Charles Darwin established evolution by common descent
as the dominant scientific explanation of biological diversity.
This hedgehog has no pigmentation due to a mutation.
Mutations are permanent, transmissible changes to
the genetic material (DNA or RNA) of a cell or virus.
Mutations result from errors in DNA replication during cell
division and by exposure to radiation

Principles
Protein evolution is based on study of two principle
Population genetics
Evolutionary biology
 if the protein accumulates a destabilizing mutation which
compromises its ability to function, natural selection will
rapidly remove it
Protein duplication.

Continue..
Recombination is a process that results in
genetic exchange between chromosomes or
chromosomal regions
Gene conversion is a type of recombination
that is the product of DNA repair where
nucleotide damage is corrected using an
homologous genomic region as a template

Genetic drift is the change of allele frequencies from one
generation to the next due to stochastic effects
of random sampling in finite populations.
Rate of evolution
Histone H4 shows extreme conservation; it has essentially
the same sequence in all eukaryotes examined.
functions of H4 are extremely dependent on its entire struc
ture.

Levels of protein organization

Overview of Protein Structure
Primary Structure - The sequence of amino acids in
the polypeptide chain
Secondary Structure - The formation of α helices and
β pleated sheets due to hydrogen bonding between the
peptide backbone
Tertiary Structure - Folding of helices and sheets
influenced by R group bonding
Quaternary Structure - The association of more than
one polypeptide into a protein complex influenced by R
group bonding

Primary Structure
The sequence of amino acids in the primary structure determines
the folding of the molecule.

Peptide Bond
 Two or more amino acids are joined by condensation reaction
i.e, removal of water molecule forms peptide bond
 The partial double-bond character of the peptide bond defines
the conformations a polypeptide chain may assume.
 It is shorter then a single bond
 Rigid & planar.

Protein Secondary Structure
• The primary sequence must organize itself to form a
compact structure. This is done in an elegant fashion
by forming secondary structure elements.
The peptide backbone has areas of positive charge
and negative charge
These areas can interact with one another to form
hydrogen bonds
The result of these hydrogen bonds are two types of
structures:
α- helices
β- pleated sheets

Alpha Helix
It is the most common confirmation.
It is a spiral structure.
Tightly packed coiled polypeptide backbone, with
extending side chains, R groups protrude outward
from the helical back bone.
Height of single turn of helix is 5.4Å with 3.6 amino
acid residue.
Right-handed α-helix predominates in nature.
It is stabilized by H-bonding between amide
hydrogens and carbonyl oxygens of peptide bonds.

β- pleated sheets
The backbone of the polypeptide chain is
extended into a zigzag rather than helical
structure.
The zigzag polypeptide chains can be arranged
side by side, hydrogen bonds are formed
between adjacent segments.
The adjacent sheets can be either parallel or
antiparallel

Tertiary Structure
 Folding of helices and sheets influenced by R group bonding
classified as
1. Fibrous Protein- chains arranged to form long strands and
usually consist largely of a single type of secondary structure.
Structures that provide support, shape, and external
protection to vertebrates are made of fibrous proteins.
2. Globular protein- chains arranged to form spherical shapes
and often contain several types of secondary structures.
Enzymes are mostly globular in nature.

Collagen
structure of collagen

Tertiary Structure
Factors influencing tertiary structure include:
Hydrophobic interactions
Hydrogen bonding
Disulphide bridges
Ionic bonds

Quaternary structure
 Quaternary structure results from the interaction of
independent polypeptide chains
Factors influencing quaternary structure include:
 Hydrophobic interactions
 Hydrogen bonding
 The shape and charge distribution on amino acids of
associating polypeptides.
e.g., Haemoglobin made up of
4 polypeptide chains.

Protein stability
Presented by Amna shehzadi

Protein stability
Protein stability is the net balance of forces, which
determine whether a protein will be in its native folded
conformation or a denatured state.
Protein stability normally refers to the physical
(thermodynamic) stability, not the chemical stability.

Forces involve in protein stabilization
Hydrogen Bonding.
Vander Waals interactions.
Ionic strengths.
Disulfide bonds
Hydrophobicity: the dominant force in protein folding Forces involved in Protein
stabilization

Factor affecting protein stability
 Temperature.
Extreme temperature make protein unstable.
 pH
Extreme pH cause unstabilty in protein.
 Organic Solvent.
 Unstable the protein
 Chaotropic agent.
 Urea and guanidinium hydrochloride.
 Destroy the tertiary structure.

Disulfide bond
If their disulfides are broken (i.e. reduced) and then
carboxymethylated with iodoacetate, the resulting protein is
denatured, i.e. unfolded, or mostly unfolded
Ligand binding
Ligand binding increases the stability of the protein.

Protein denaturation
 A loss of three-dimensional structure sufficient to cause loss
of function is called denaturation
 Alterations in the environment (pH, salt concentration,
temperature etc.) disrupt bonds and forces of attraction.

Protein folding
A folding protein follows multiple pathways
from high energy and high Entropy to low
energy and low entropy.
Amyloid diseases result from protein
misfolding.

Levinthal paradox
 A polypeptide chain of 101 amino acid residues would have to
sample 3 100 = 5 × 10 47 conformations, if each bond
connecting two consecutive residues has only three possible
configurations. If the sampling takes place at a rate equal to that
of bond vibrations, then it would take 10 27 years for an unfolded
polypeptide chain to complete the search for its native
conformation.
 The discrepancy between this large time estimate and the real
folding times of proteins, which are in the seconds timescale or
faster, is commonly referred to as the Levinthal paradox

Energy
landscape model
all folding protein
molecules are guided
by an energy bias to
traverse an energy
landscape towards the
native conformation.
Energy landscape
view of protein folding

Factor of Protein misfolding
Absence of normal supporting/co factors
Absence of chaperone protein
Change in temp and pH

Factors of protein misfolding
Mutations Premature termination of Translation
Fault in post-translational modifications
Strong Promoters
High Inducer concentrations Reasons for protein misfolding
Loss of conformation due to stress

Chaperon
These are protein molecule
Asist in protein folding.
Prevent aggregation.
Examples
GroEL is bacterial chaperone
HSP in eukaryotes

Anfinsen experiment –Spontaneous folding
Ribonuclease is a small protein that contain 8 cysteine linked
via four disulfide bond
Urea in presence of 2- mercaptoethanol fully denature
ribonuclease.
When urea and 2 mercaptoethanol are removed the protein
spontaneously refolds and correct disulfide bonds are
formed.
The sequence alone determines the native conformation.
Awarded Nobel prize in 1972.

Renaturation
Native structure and biological activity of some
globular proteins can be regained if the
denaturing agent will be removed.
Ribonuclease present a classical example of
renaturation.

Function of protein
Storage and transport

Storage proteins:-
1. Plant accumulate storage substances such as
starch lipid and proteins in certain phases of
development .
Storage proteins accumulate in both vegetative and
reproductive tissues and serve as a reservoir to be
used in later stages of plant development

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The accumulation of storage protein is thus
beneficial for the survival of plants.
Storage proteins are also an important source of
dietary plant proteins.

Storage protein in wheat:-
The two major groups of storage protein in the
endosperm of wheat such as:
gliadins
and glutenins

Ferritin as a storage protein
 Ferritin is a protein that stores iron and releases it in
a controlled fashion.
 The body has a buffer against iron deficiency (if the
blood has too little iron, ferritin can releases more).
Ferritin can help to store the excess iron.

Facilitated diffusion
 Large molecules or those with a charge (not soluble in lipids) need to help of a
protein to pass across a cell membrane.
 Protein from a channel and molecules move through the doorway (from high to low
concentration ).
 Each channel is specific to a particuler type of molecule.

Active transport
Active transport also relies on transport proteins and
is able to transport substances against a
concentration gradient. This is
because cellular energy (ATP) is proteins,then ,play
an integral role in the function of a cell.

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Many are embedded in the cell,s membranes or
span the entire lipid bilayer where they play an
important role in recognition signaling,and transport.

Hemoglobin
 Hemoglobin is the protein that makes blood red.
It is composed of four protein chains . Two
alpha chains and two beta chains. Each with a
ring-like heme group containing an iron atom.

Continue…
Oxygen binds reversibly to these iron atoms and is
transported through blood.


Proteins Function In Defense System
and Cell Signaling

Defensive Proteins
 Defensive proteins are better known as antibodies.
 These are a key part of the immune system.
 Antibodies are formed in the white blood cells and
fight off infections and viruses.

Defensive Proteins
 Defense proteins such as antibodies, which are also known
as immunoglobulin.
 Any of a class of proteins present in the serum and cells of
the immune system, which function as antibodies =
immunoglobulin.
 The antibodies set off a reaction inside the human body
that works to fight off the offending organism.

Defensive Proteins
 Often, people believe the antibodies actually destroy
bacteria
 But the direct reaction is usually more along the lines of
rendering the invading organism harmless.
 Example of defensive proteins is Salivary Defense Proteins.

Salivary Defense Proteins
Saliva is a body fluid, secreted by three pairs of major
salivary glands (parotid, submandibular and
sublingual).
Parotid
 Major salivary gland.
 In humans, the two parotid glands are present.
 The word parotid mean “beside the ear”

 Submandibular
 Located beneath the floor of the mouth.
 They each weigh about 15 grams and contribute some 60–67%
of unstimulated saliva secretion.
 Sublingual
 They are the smallest, most diffuse.
 They provide only 3-5% of the total salivary volume.

 There are numerous defense proteins present in the saliva.
 Some of these defense proteins, like salivary
immunoglobulin's, and salivary chaperokine HSP70/HSPA,
are involved in both innate and acquired immune
activation.
 Salivary chaperokine HSP70 = Salivary enzymes that are
major role in defense system.

 Salivary cationic peptides and other salivary defense
proteins, like
 Lysozyme, BPI, BPI-like, PLUNC proteins, salivary amylase,
Cystatins, prolin-rich proteins, Mucins, Peroxidases,
statherin
 Are primarily responsible for innate immunity
 And these all are proteins that present in saliva naturally.

Mechanism Of Salivary Defense Proteins
 Five primary defense networks of salivary proteins in
whole saliva.
 First
 The first network may be responsible for microbial
agglutination.
 This network may include those salivary proteins and
peptides which bind bacteria (microbes).

 Second
 The second network responsible for lysis of microbial membranes.
 This network primarily targets bacteria, and are likely to include salivary
cationic peptides and lysozyme.
 3rd and 4th
 The third and fourth networks responsible for antifungal and antiviral
properties of the saliva respectively.
 These networks may include numerous salivary proteins exerting
various antifungal or antiviral properties respectively.

 5th
 Responsible for immune regulatory.
 This network is likely to include all those salivary proteins
which exert immune activator/modulator properties.
 This network may be important for the fine-regulation of
the local action of the mucosal immune system.

Cell Signaling
 Cells typically communicate using chemical signals.
 These chemical signals, which are proteins or other
molecules produced by a sending cell,
 Often secreted from the cell and released into the
extracellular space.
 There, they can float – like messages in a bottle – over to
neighboring cells.

Cell Signaling
 Not all cells can “hear” a particular chemical message.
 In order to detect a signal, a neighbor cell must have the
right receptor for that signal.
 When a signaling molecule binds to its receptor, it alters
the shape or activity of the receptor
 Triggering a change inside of the cell.
 Signaling molecules are often called ligands

Protein purification:-
Protein purification is a technique by which a single
protein type is isolated from a complex mixture such
as a cell lysate.
It can refer to purification of a native protein from a
biological sample or of a recombinant protein

Methods of protein purification
(1) Extraction
(2) Precipitation
(3) Ultracentrifugation
(4)Chromatographic Methods.

1- Extraction:-
1. First the protein brought into
solution.
2. By freezing and thawing,
sonication, homogenization by
high pressure or
permeabilization by organic
solvents.
3. Then soluble protein will be in
the solvent.
4. After that protein is separated
by centrifugation.

2-Precipitation:-
In bulk protein purification.
The first proteins to be purified are water-soluble
proteins.
Sodium dodecyl sulphate (SDS) can be used to
dissolve cell membranes.

Procedure :-
1-
• isolate proteins is precipitation with
ammonium sulphate (NH4)2SO4.
2-
• By adding increasing amounts of ammonium
sulphate
3-
• collecting the different fractions of precipitate
protein.

3- Ultracentrifugation
When a vessel containing
a mixture of proteins or
other, is rotated at high
speed the angular
momentum yields an
outward force to each
particle that is
proportional to its mass.

Procedure :-
Suspensions of particles are “spun”
in a centrifuge, a “pellet” may form
at the bottom of the vessel.
The remaining particles still
remaining mostly in the liquid are
called the “supernatant”
Supernatant is removed from the
vessel.

4-chromatographic methods:-
Procedure :-
The basic procedure in chromatography is to
flow the solution containing the protein through
a column packed with various materials.

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Different proteins can thus be separated by the
time required to pass the column, or the conditions
required to elute the protein from the column.
 Usually proteins are detected as they are coming
off the column by their absorbance at 280 nm.

Continue…
There are various chromatographic
techniques used for the purification of
proteins.
Size exclusion chromatography.
Ion exchange chromography.
Affinity chromatography.
Immunoaffinity chromatography.
HPLC.

Size exclusion chromatography:-
Chromatography can be used to separate
protein in solution or denaturing conditions by
using porous gels. This technique is known as
size exclusion chromatography.

Principle
Smaller molecules have to traverse a larger
volume in a porous matrix.Proteins of a certain
range in size will require a variable volume of
eluant (solvent) before being collected at the
other end of the column of gel.

Procedure
1.The eluent is usually pooled in different test tubes.
2.All test tubes containing no measurable trace of the
protein to purify are discarded.
3.The remaining solution is thus made of the protein to
purify.

Protein structure
determination

Structure determination:
Determining 3D structure of protein help us to understand
mechanism of action of protein and it’s functions.
That:• How proteins interact with other molecules ? • How
they perform catalysis in the case of enzymes ? •
Interaction of protein with other molecules including
protein itself. • Miscoding and/or misfolding of proteins
associated with diseases.
3 methods used for structure determination.

X-Ray Crystallography:
 A form of very high resolution microscopy.
 Enables us to visualize protein structures at the atomic level
 Enhances our understanding of protein function.
• Principle :
– It is based on the fact that X-rays are diffracted by crystals.
Direct detection of atoms position in crystals

Why we use X-rays and crystals?
• X-rays is in the order of atom diameter and bond
lengths, allowing these to be individually resolved.
• No lenses available to focus X-rays. Crystal acts as a
magnifier of the scattering of X-rays.

Steps for determining protein structure:
1. Protein
purification.
2. Protein
crystallization.
3. Data
collection.
4. Structure
Solution
(Phasing)
5. Structure
determination
(Model
building and
refinement)

Step1:Protein purification:
series of processes intended to isolate one or a few
proteins from a complex mixture, usually cells,
tissues or whole organisms.
 Characterization of the function.
 Structure of the protein

Step2:Protein crystallization:
 X-ray scattering from a single unit would be unimaginably weak.
 A crystal arranges a huge number of molecules in the same orientation.
 Scattered waves add up in phase and increase Signal to a level which can be
measured-structure determinations.
 Protein crystallization Crystals MUST be: Small in size
 Less than 1 millimeter PERFECT
 No cracks

Step3:Data collection:
 Mounting of crystals
 Exposing x rays
 The scattered X-rays are captured as a diffraction
pattern on a detector such as film or an electronic
device
 Rotate crystal through 1 degree and Record XRD
pattern
 If XRD pattern is very crowded, reduce the
degree of rotation.

Step4:Structure solution(phasing)
 In order to visualize our structure we need to solve the phase
problem.
 If we already have the coordinates of a similar protein we can
try to solve the structure using a process called Molecular
Replacement which involves taking this model and rotating and
translating it into our new crystal system until we get a good
match to our experimental data.

Step5:Structure determination:
 Fitting of protein sequence.
 Automated improvement of the model, so
it explains the observed data better.

Nuclear Magnetic Resonance
Spectroscopy
Presented by: Afifa khizer

Protein structure determination: NMR
 NMR is a spectroscopy technique which is based on the absorption
of electromagnetic radiation by nuclei of the atoms.
 Proton nuclear magnetic resonance spectroscopy is one of the most
powerful tools for elucidating the number of hydrogen or proton in
the compound.
 It is used to study a wide variety of nuclei:
1H
13C
15N
19F
31P

Aim:
measure set of distances between atomic nuclei.
Why?
– For proteins that are hard to crystallize.
– For proteins that can be dissolved at high
concentrations.
– To study dynamics of the protein:
conformational equilibria, folding and intra-,
intermolecular interactions.

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.

Steps:
1.Protein solution
2.NMR spectroscopy
3.Sequencial resonance assignment
4.Conformational constraints collection
5.Structure calculation

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.

4.Collection of conformational constraints:
Geometric conformational information derived
from NMR.. 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.

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.
Biological specimen:
1.Thin film.
2.Vitreous section.

Protein stability

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