We often think of proteins as nutrients in the food we eat or the main component of muscles,

but proteins are also microscopic molecules inside of cells that perform diverse and vital jobs.
Proteins are the main molecular machines in our bodies. They perform a wide range of functions,
from digesting and processing nutrients, converting energy and aiding cell structure to transmitting
signals in cells and the whole body. In order to perform these highly specific functions, proteins have
to adopt a well-defined, three-dimensional structure. Remarkably, in most cases they find this
structure unaided once they have been formed out of their individual building blocks, amino acids, as
a long chain molecule in the cell. A protein’s function depends on its shape, and when protein
formation goes awry, the resulting misshapen proteins cause problems that range from bad, when
proteins neglect their important work, to ugly, when they form a sticky, clumpy mess inside of cells.
Protein formation is an error-prone process, and mistakes along the way have been linked to a number
of human diseases. Proteins are the workhorses of the cell. Each expertly performs a specific task.
Others bind to specific molecules and shuttle them to new locations, and still others catalyze reactions
that allow cells to divide and grow. This wealth of diversity and specificity in function is made
possible by a seemingly simple property of proteins: they fold. Protein folding is a process by which a
polypeptide chain folds to become a biologically active protein in its native 3D structure. Protein
structure is crucial to its function. Folded proteins are held together by various molecular interactions.
During translation, each protein is synthesized as a linear chain of amino acids or a random coil which
does not have a stable 3D structure. The amino acids in the chain eventually interact with each other
to form a well-defined, folded protein. The amino acid sequence of a protein determines its 3D
structure. Folding of proteins into their correct native structure is the key to their function. However,
failure to fold properly produces inactive or toxic proteins that malfunction and cause a number of
diseases. The process of protein folding can also go wrong, which means the proteins affected are no
longer able to perform their function. In some cases, this can even have much more serious
consequences if these misfolded proteins clump and trigger neurodegenerative diseases such as
Alzheimer's or Parkinson's disease. In the course of evolution, a crucial factor in the development of
proteins has thus been to avoid such "misfolding processes." However, this is no easy task since the
same molecular interactions that stabilize the correct structure of the individual proteins can also bring
about interactions between protein molecules, causing them to misfold ( “Similarities cause..”,
2011).
Current advances in medicine and technology are making our lives longer. Sadly, as our life
expectancy increases, the chances of getting a degenerative disease like Alzheimer's or Parkinson's
also increases. Why is this? As incredible as it might sound, these diseases are caused not
by bacteria or viruses but rather by something conceptually quite simple: incorrect protein folding.
Since proteins are involved in a multitude of processes in the human body, misfolding is potentially
harmful. The chemicals often fold correctly, but this isn’t always the case. A variety of environmental
factors surrounding a protein can affect its final shape. These factors include the local pH and
temperature and the chemical composition of proteins located close to the one being folded. Gene
mutations can also affect folding by changing the structure of a protein. In young people or in healthy
cells, altered and misfolded proteins are often broken down and removed by the cell and no damage is
done. In older people or in people with certain genetic problems, the number of misfolded proteins
may overwhelm the cell's ability to remove them. Under these conditions, the damaged molecules
tend to clump together. Alzheimer's disease is that the commonest reason behind dementia. It is a
very unpleasant neurodegenerative condition. An affected person gradually develops severe memory
loss, difficulty in solving problems and making decisions, confusion, and major changes in
personality and behavior. The disease is characterized by tangles of misfolded beta-amyloid proteins
(or more accurately, protein fragments) within the brain. These tangles form around the nerve cells, or
neurons, and are called plaques. A second brain protein called tau also becomes misfolded and tangled
during Alzheimer's. Tau tangles form inside the neurons. The misfolded proteins have altered
properties and can't function properly. Brain neurons die and therefore the patient develops
progressive memory loss and behaviour problems. Parkinson's disease is another neurodegenerative
condition. In this illness, dopamine-secreting cells in a part of the brain called the substantia nigra die
and the patient develops movement problems. Dopamine is a neurotransmitter, which is a chemical
that transmits a signal from one neuron to another. Another feature of Parkinson's disease is the
appearance of small clumps of misfolded proteins inside neurons in the substantia nigra. The clumps
are known as Lewy bodies and are made of a protein called alpha-synuclein. As in Alzheimer's
disease, the misfolding causes the altered proteins in the brain to aggregate ( Crampton, 2019).
2. A protein must be purified before its structure and the mechanism of its action can be studied.
However, because proteins vary in size, charge, and water solubility, no single method can be used to
isolate all proteins. Although the sequence of amino acids in a protein uniquely determines its
function, the most useful physical characteristic for separation of proteins is size, defined as either
length or mass. In this section, we briefly outline different techniques for separating proteins based on
their size and other properties. These techniques also apply to the separation of nucleic acids and other
biomolecules. We then consider general methods for detecting, or assaying, specific proteins, for
tracking biological activity. Finally, we discuss several techniques for characterizing a protein’s mass,
sequence, and three-dimensional structure.
Extraction of Proteins from Cells
At a concentration higher than its critical micelle
concentration (CMC), a detergent solubilizes lipids and
integral membrane proteins, forming mixed micelles containing
detergent, protein, and lipid molecules. At concentrations below
the CMC, many detergents (e.g., octylglucoside) can dissolve
membrane proteins without forming micelles by coating the
membrane-spanning regions. Since octylglucoside has a high
CMC, it is particularly effective in solubilizing integral
membrane proteins without denaturing them or forming mixed
micelles.
The most common initial step in protein purification is
separation of soluble proteins from insoluble cellular material
by differential centrifugation. The centrifugal force and duration of
centrifugation are adjusted to ensure that the insoluble materials
sediment into a pellet. After a starting mixture of a cell homogenate
is poured into a tube and spun in a centrifuge, cell organelles such
as nuclei collect into a pellet, but the soluble proteins remain in the
supernatant. The supernatant fraction still contains a large mixture
of proteins, which can be collected by decanting the supernatant
and then subjecting it to further purification methods. Differential
centrifugation separates a mixture of particles (macromolecules,
cell organ-elles, and cells) that differ in mass or density. The most
dense particles collect at the bottom of the tube as a pellet. The
least dense particles remain in the liquid supernatant, which can be
transferred to another tube. Rate-zonal centrifugation separates
particles or molecules that differ in mass but may be similar in
shape and density. Here two particles of different mass separate
into two zones.

Column Chromatography
Column chromatography is a technique which is used to
separate a single chemical compound from a mixture dissolved in
a fluid. It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move through the
column at different rates which allow them to get separated in
fractions. This technique can be used on small scale as well as
large scale to purify materials that can be used in future experiments. This method is a type
of adsorption chromatography technique.

Size – Exclusion Chromatography

Size exclusion chromatography (SEC), also known as gel
filtration, is the mildest of all the chromatography techniques. Gel
filtration chromatography separates proteins that differ in size. A
mixture of proteins is carefully layered on the top of a glass
cylinder packed with porous beads. Smaller proteins travel
through the column more slowly than larger proteins. Thus
different proteins have different elution volumes and can be
collected in separate liquid fractions from the bottom.
Affinity Chromatography
 In antibody-affinity chromatography, a specific antibody
is covalently attached to beads packed in a column. Only protein
with high affinity for the antibody is retained by the column; all
the nonbinding proteins flow through. The bound protein is eluted
with an acidic solution, which disrupts the antigen-antibody
complexes.
Ion – Exchange Chromatography
Ion-exchange chromatography separates proteins that
differ in net charge in columns packed with special beads that
carry either a positive charge (shown here) or a negative charge.
Proteins having the same net charge as the beads are repelled and
flow through the column, whereas proteins having the opposite
charge bind to the beads. Bound proteins, in this case negatively
charged, are eluted by passing a salt gradient (usually of NaCl or
KCl) through the column. As the ions bind to the beads, they
desorb the protein.

Electrophoresis
Electrophoresis is a technique for separating, or resolving, molecules in a mixture under the
influence of an applied electric field. Dissolved molecules in an electric field move, or migrate, at a
speed determined by their charge: mass ratio. For example, if two
molecules have the same mass and shape, the one with the greater
net charge will move faster toward an electrode. The separation of
small molecules, such as amino acids and nucleotides, is one of
the many uses of electrophoresis. In this case, a small drop of
sample is deposited on a strip of filter paper or other
porous substrate, which is then soaked with a conducting solution.
When an electric field is applied at the ends of the strip, small
molecules dissolved in the conducting solution move along the
strip at a rate corresponding to the magnitude of their charge.

SDS – polyacrylamide Gel Electrophoresis (SDS – PAGE)

SDS-polyacrylamide gel electrophoresis, a common
technique for separating proteins at good resolution.
The protein mixture first is treated with SDS, a negatively charged
detergent that binds to proteins. This binding
dissociates multimeric proteins and forces all polypeptide chains into
dena-tured conformations with nearly identical charge:mass ratios.
During electrophoresis, the SDS-protein complexes migrate through the polyacrylamide gel. Small
proteins are able to move through the pores more easily, and faster, than larger proteins. Thus the
proteins separate into bands according to their size as they migrate through the gel. The separated
protein bands are visualized by staining with a dye.

Isoelectric Focusing
Isoelectric focusing (IEF) is one of the most commonly used
techniques for the separation of proteins. IEF separations are based on
the pH dependence of the electrophoretic mobilities of the protein
molecules. Isoelectric focusing makes use of electrical charge
properties of molecules to focus them in defined zones in a separation
medium. It is the focusing mechanism that distinguishes IEF from
other separation processes and makes it unique among the separation
methods. In sharp contrast, the basic separation mechanism of IEF
imposes forces on the molecules that directly counteract the
dispersive effects of diffusion. During the separation process, the
molecules in the sample accumulate in specific and predictable
locations in the medium, regardless of their initial distribution. This
focusing mechanism also distinguishes IEF from various modes of
electrophoresis. It is important to note that IEF is a high-resolution
method. It is well suited for both analytical and preparative
applications.

Determining the Primary Structure of Proteins

The classic method for determining the amino acid sequence
of a protein involves Edman degradation . In this procedure the amino
group at the N-terminus of a polypeptide is labeled and its amino acid
then cleaved from the polypeptide and identified by high-pressure
liquid chromatography. The polypeptide is left one residue shorter,
with a new amino acid at the N-terminus. The cycle is repeated on the
ever shortening polypeptide until all the residues have been identified.
Chemical determination of the sequence of a protein by Edman
degradation, which involves a repetitive three-step procedureIn the
first step, the polypeptide Nterminus is reacted with
phenylisothiocyanate (PITC). In the second step, the N-
terminal amino acid is cleaved from the polypeptide by
acid hydrolysis, yielding the cyclic phenylthiohydantoin (PTH)
derivative and a polypeptide that is shorter at its Nterminus by one
residue. These two steps are then repeated with the shortened
polypeptide. The PTH derivative formed in each cycle is identified by
liquid chromatography.

3. When is a life form not a life form? When it’s a virus. Viruses are strange things that straddle the
fence between living and non-living. On the one hand, if they’re floating around in the air or sticking
on a doorknob, they’re inert. They’re about as alive as a rock. But if they come into contact with a
suitable plant, animal or bacterial cell, they spring into action. They infect and take over the cell like
pirates hijacking a ship. A virus is basically a tiny bundle of genetic material either DNA or RNA
carried in a shell called the vral coat, or capsid, which is made up of bits of protein called capsomeres.
Some viruses have an additional layer around this coat called an envelope. Tha’s basically all there to
viruses. When viruses come into contact with host cells, they trigger the cells to engulf them, or fuse
themselves to the cell membrane so they can release their DNA into the cell. Once inside a host cell,
viruses take over its machinery to reproduce. Viruses override the host’s cells normal functioning with
their own set of instructions that shut down the production of host proteins and direct the cell to
produce viral proteins to make new virus particles that will invade new hosts. Some viruses, inert their
genetic material into the host cell’s DNA, where they begin directing the copying of their genes or
simply lie dormant for years or a lifetime. Either way, the host cell does all the actual work while the
viruses simply provide the instructions. But how do viruses differ to a living cell? Like living
organisms, viruses contain genetic instructions. However, they lack the machinery needed to carry out
these instruction. On their own, they are inert chemicals and cannot perform any life functions.
However, if they enter a living host cell and they will use the cell’s machinery to replicate. Cells are
the basic units of life, and in addition to the single celled bacteria growing on your table, cells also
make up all the tissues and organs in our bodies. Even smaller than cells are viruses. Unlike
cells, viruses are non-living (arguably) infectious particles. Although there are also differences in
structure, size, and life cycle. Genetic material is the instructions for all cell function. In cells, the
genetic material is deoxyribonucleic acid (or DNA). DNA is made of individual pieces
called nucleotides that are strung together in two long chains that twist together, forming a double
helix. However, viruses break the rules a little bit. Viruses can use DNA or RNA as their genetic
material. The DNA and RNA don’t even have to have the same structure as they do in normal cells.
Cells have double stranded DNA molecule and many strands of single stranded RNA as the copies.
Viruses, however, can have double stranded DNA, single stranded DNA, double stranded RNA , or
single stranded RNA. They convert RNA to DNA and the back to RNA to make proteins , which does
not happen in the cells.

An example of a virus with a single stranded RNA genome is human immunodeficiency virus
(HIV). This sexually or intravenously transmitted virus infects a type of immune cell called T-cell.
When HIV infects the T-cells, it kills them, weakening the patient’s immune system and making them
more susceptible to infections. HIV is a sexually transmitted infection (STI). Human
immunodeficiency virus (HIV) is a virus that attacks immune cells called CD4 cells, which are a type
of T cell. These are white blood cells that move around the body, detecting faults and anomalies in
cells as well as infections. When HIV targets and infiltrates these cells, it reduces the body’s ability to
combat other diseases. It can also be spread by contact with infected blood or from mother to child
during pregnancy, childbirth or breast-feeding. HIV is a virus that targets and alters the immune
system, increasing the risk and impact of other infections and diseases. Without treatment, the
infection might progress to an advanced disease stage called AIDS. AIDS is the most advanced stage
of HIV infection. Once HIV infection develops into AIDS, infections and cancer pose a greater risk.
Without treatment, HIV infection is likely to develop into AIDS as the immune system gradually
wears down (Felman, 2018). Another virus which is very rampant until now is the coronavirus disease
2019 (COVID-19), which used to be called the novel coronavirus (2019-nCoV), which is a new type
of coronavirus. It causes respiratory illness in people. It was first identified in Wuhan, China.
Coronavirus disease 2019 (COVID-19) is defined as illness caused by a novel coronavirus now called
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; formerly called 2019-nCoV), which
was first identified amid an outbreak of respiratory illness cases in Wuhan City, Hubei Province,
China. It was initially reported to the WHO on December 31, 2019. On January 30, 2020, the WHO
declared the COVID-19 outbreak a global health emergency.   Illness caused by SARS-CoV-2 was
recently termed COVID-19 by the WHO, the new acronym derived from "coronavirus disease 2019."
The name was chosen to avoid stigmatizing the virus's origins in terms of populations, geography, or
animal associations. Coronaviruses are a group of common viruses. They are named for the crown-
like spikes on the surface of the virus. They are single-stranded RNA viruses that have a lipid
envelope studded with club-shaped projections, infect birds and many mammals including humans,
and include the causative agents of MERS and SARS Some coronaviruses only affect animals, but
others can also affect humans. Most people get infected with human coronaviruses at some time in
their life. They usually cause mild to moderate upper-respiratory infections, like the common cold.
But they can also cause more severe illnesses such as bronchitis and pneumonia (“Coronaviurs
Infections”, 2020).

References:

Crampton, L. (2019, October 15). Misfolded Proteins in Alzheimer's and Parkinson's Diseases.
Retrieved March 1, 2020, from https://owlcation.com/stem/Misfolded-Proteins-Human-Diseases-and-
Possible-Treatments

Coronavirus Infections | Coronavirus. (2020, February 27). Retrieved March 1, 2020, from
https://medlineplus.gov/coronavirusinfections.html

Felman, A. (2018, November 29). HIV and AIDS: Overview, causes, symptoms, and treatments.
Retrieved March 1, 2020, from https://www.medicalnewstoday.com/articles/17131#what-is-aids

Lodish, H. (1970, January 1). Purifying, Detecting, and Characterizing Proteins. Retrieved March 1,
2020, from https://www.ncbi.nlm.nih.gov/books/NBK21589/

Similarities cause protein misfolding. (2011, June 4). Retrieved March 1, 2020, from
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misfolding. (2011, June 4). Retrieved March 1, 2020, from
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