1. Explain and illustrate the primary, tertiary and quaternary structure of protein.

Proteins are biological polymers composed of amino acids. Amino acids, linked together by peptide bonds, form a
polypeptide chain. One or more polypeptide chains twisted into a 3-D shape form a protein. Proteins have complex
shapes that include various folds, loops, and curves. Folding in proteins happens spontaneously. Chemical bonding
between portions of the polypeptide chain aid in holding the protein together and giving it its shape. There are two
general classes of protein molecules: globular proteins and fibrous proteins. Globular proteins are generally
compact, soluble, and spherical in shape. Fibrous proteins are typically elongated and insoluble. Globular and
fibrous proteins may exhibit one or more of four types of protein structure. The four levels of protein structure are
distinguished from one another by the degree of complexity in the polypeptide chain. A single protein molecule may
contain one or more of the protein structure types: primary, secondary, tertiary, and quaternary structure.

 Primary Structure describes the unique order in which amino acids are linked together to form a protein.
Proteins are constructed from a set of 20 amino acids. Generally, amino acids have the following structural
properties:

 A carbon (the alpha carbon) bonded to the four groups below:

 A hydrogen atom (H)
 A Carboxyl group (-COOH)
 An Amino group (-NH2)
 A "variable" group or "R" group

All amino acids have the alpha carbon bonded to a hydrogen atom, carboxyl group, and an amino group. The "R"
group varies among amino acids and determines the differences between these protein monomers. The amino acid
sequence of a protein is determined by the information found in the cellular genetic code. The order of amino acids
in a polypeptide chain is unique and specific to a particular protein. Altering a single amino acid causes a gene
mutation, which most often results in a non-functioning protein.

 Secondary Structure refers to the coiling or folding of a polypeptide chain that gives the protein its 3-D
shape. There are two types of secondary structures observed in proteins. One type is the alpha (α)
helix structure. This structure resembles a coiled spring and is secured by hydrogen bonding in the
polypeptide chain. The second type of secondary structure in proteins is the beta (β) pleated sheet. This
structure appears to be folded or pleated and is held together by hydrogen bonding between polypeptide
units of the folded chain that lie adjacent to one another.
 Tertiary Structure refers to the comprehensive 3-D structure of the polypeptide chain of a protein. There
are several types of bonds and forces that hold a protein in its tertiary structure.

 Hydrophobic interactions greatly contribute to the folding and shaping of a protein. The "R" group of the
amino acid is either hydrophobic or hydrophilic. The amino acids with hydrophilic "R" groups will seek
contact with their aqueous environment, while amino acids with hydrophobic "R" groups will seek to avoid
water and position themselves towards the center of the protein.
 Hydrogen bonding in the polypeptide chain and between amino acid "R" groups helps to stabilize protein
structure by holding the protein in the shape established by the hydrophobic interactions.
 Due to protein folding, ionic bonding can occur between the positively and negatively charged "R" groups
that come in close contact with one another.
 Folding can also result in covalent bonding between the "R" groups of cysteine amino acids. This type of
bonding forms what is called a disulfide bridge. Interactions called van der Waals forces also assist in the
stabilization of protein structure. These interactions pertain to the attractive and repulsive forces that occur
between molecules that become polarized. These forces contribute to the bonding that occurs between
molecules.

 Quaternary Structure refers to the structure of a protein macromolecule formed by interactions between
multiple polypeptide chains. Each polypeptide chain is referred to as a subunit. Proteins with quaternary
structure may consist of more than one of the same type of protein subunit. They may also be composed of
different subunits. Hemoglobin is an example of a protein with quaternary structure. Hemoglobin, found in
the blood, is an iron-containing protein that binds oxygen molecules. It contains four subunits: two alpha
subunits and two beta subunits.

2. Identify the major recognized types of secondary structure and explain

supersecondary motifs.
 Types of Secondary Structure. There are three common secondary structures in proteins, namely alpha
helices, beta sheets, and turns. That which cannot be classified as one of the standard three classes is
usually grouped into a category called "other" or "random coil".
3. Describe the kind and relative strenghths of the forces that stabilize each other of protein
structure.Protein structures are governed primarily by hydrophobic effects and by interactions between polar
residues and other types of bonds. The hydrophobic effect is the major determination of
original protein structure. The aggregation of nonpolar side chains in the interior of a protein is favored by the
increase in Entropy of the water molecules that would otherwise form cages around the hydrophobic groups.
Hydrophobic side chains give a good indication as to which portions of a polypeptide chain are inside, out of
contact with the aqueous solvent. Hydrogen bonding is a central feature in protein structure but only make
minor contributions to protein stability. Hydrogen bonds fine tune the tertiary structure by selecting the unique
structure of a protein from among a relatively small number of hydrophobically stabilized conformations.
Disulfide bonding can form within and between polypeptide chains as proteins fold to its native conformation.
Metal ions may also function to internally cross link proteins.

4. Identify the physiologic roles in protein maturation of chaperones, protein disulfide isomerase and
peptidyl proline cis-trans isomerase
a.) Maturation of chaperones- Molecular chaperones are able to promote the efficient folding of proteins and
prevent aggregation by providing a protected and privileged folding environment within the cell. To
understand the mechanism by which chaperones assist in protein maturation and maintaining protein
homeostasis (proteostasis), one must understand: how they recognize proteins, how their binding cycle is
maintained, which cofactors are involved, and how these cofactors assist in substrate selection and the
regulation of the chaperone-binding cycle.
b.) Protein disulfide- Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox
proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and
redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and
subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide
an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as
well as its physiological and pathophysiological functions related to redox regulation and protein folding.

5. Configuration the set of possible shapes a molecule can have by means of rotation about single bonds only.
Configuration is the relative position of the atoms in a molecule that can be changed exclusively by cleaving
and forming new chemical bonds.

6. Conformation- The spatial arrangement of the atoms affording distinction between stereoisomers which can be
interconverted by rotations about formally single bonds.
Some authorities extend the term to include inversion at trigonal pyramidal centres and other polytopal
rearrangements.

7. X-ray Crystallography - is a technique used for determining the atomic and molecular structure of a crystal, in
which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By
measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-
dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions
of the atoms in the crystal can be determined, as well as their chemical bonds, their crystallographic disorder,
and various other information.
8. NMR spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique
used in quality control and reserach for determining the content and purity of a sample as well as its molecular
structure. For example, NMR can quantitatively analyze mixtures containing known compounds.
9. Prion disease- Prions are misfolded proteins with the ability to transmit their misfolded shape onto normal
variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in
humans and many other animals.
10. Alzheimer’s Disease- The biochemistry of Alzheimer's disease (AD), one of the most common causes of adult
dementia, is not yet very well understood. AD has been identified as a possible protein misfolding disease due
to the accumulation of abnormally folded amyloid beta protein in the brains of Alzheimer's patients.

11. Beta-thalassemias- Beta thalassemia is a blood disorder that reduces the production of hemoglobin.
Hemoglobin is the iron-containing protein in red blood cells that carries oxygen to cells throughout the body. In
people with beta thalassemia, low levels of hemoglobin lead to a lack of oxygen in many parts of the body.
 1. Describe the most important structural similarities and differences between and hemoglobin

2. Sketch the binding curves for the oxygenation of myogblin and hemoglobin

Oxygen binding to iron in the heme group pulls part of the electron density from the ferrous ion to the oxygen
molecule. It is important to leave the myoglobin in the dioxygen form rather than superoxide form when the oxygen
is released because the superoxide can be generated by itself to have a new form that gives negative effect on many
biological materials, and also the superoxide prevents the iron ion from binding to the oxygen in its ferric state
(Metmyoglobin). Superoxide and superoxide-derived oxygen species are so reactive compared to the stable O2
molecule that they could have a destructive effect both within the cell and in its environment. A distal histidine
residue in myoglobin regulates the reactivity of the heme group to make it more suitable for oxygen binding. It does
this by H-bonding with the oxygen molecule; the additional electron density of the oxygen molecule makes the H-
bond unusually strong and therefore even more effective as a stabilizing agent.An oxygen-binding curve is a plot
that shows fractional saturation versus the concentration of oxygen. By definition, fractional saturation indicates the
presence of binding sites that have oxygen. Fractional saturation can range from zero (all sites are empty) to one (all
sites are filled). The concentration of oxygen is determined by partial pressure.Hemoglobin's oxygen affinity is
relatively weak compared to myoglobin 's affinity for oxygen. Hemoglobin's oxygen-binding curve forms in the
shape of a sigmoidal curve. This is due to the cooperativity of the hemoglobin. As hemoglobin travels from the
lungs to the tissues, the pH value of its surroundings decrease, and the amount of CO2 that it reacts with increases.
Both these changes causes the hemoglobin to lose its affinity for oxygen, therefore making it drop the oxygen into
the tissues. This causes the sigmoidal curve for hemoglobin in the oxygen-binding curve and proves it's
cooperativity.

Oxygen Binding Curve (Hemoglobin vs Myoglobin) This image shows hemoglobin's oxygen binding
affinity compared with myoglobin 's affinity and the hypothetical curve that hemoglobin would have to follow if it
did not show cooperativity. In this graph, you can see hemoglobin's
sigmoidal curve, how it starts out with a little less affinity than
myoglobin, but comparable affinity to oxygen in the lungs. As the
pressure drops and the myoglobin and hemoglobin move towards the
tissues, myoglobin still maintains its high affinity for oxygen, while
hemoglobin, because of its cooperativity, suddenly loses its affinity,
therefore

making it the better transporter of oxygen than myoglobin . The gray

curve, showing no cooperativity, shows that to have the low affinity for
oxygen needed in the tissues, the hemoglobin would have started with a
smaller affinity for oxygen, therefore making it less efficient in bringing
oxygen in from the lungs.
Oxygen Binding Curve for Hemoglobin

In red blood cells, the oxygen-binding curve for hemoglobin displays an “S” shaped called a sigmoidal curve. A
sigmoidal curve shows that oxygen binding is cooperative; that is, when one site binds oxygen, the probability that
the remaining unoccupied sites that will bind to oxygen will increase.

The importance of cooperative behavior is that it allows hemoglobin to be more efficient in transporting oxygen. For
example, in the lungs, the hemoglobin is at a saturation level of 98%. However, when hemoglobin is present in the
tissues and releases oxygen, the saturation level drops to 32%; thus, 66% of the potential oxygen-binding sites are
involved in the transportation of oxygen.

Purified hemoglobin binds much more tightly to the oxygen, making it less useful in oxygen transport. The
difference in characteristics is due to the presence of 2,3-Bisphosphoglycerate(2,3-BPG) in human blood, which acts
as an allosteric effector. An allosteric effector binds in one site and affects binding in another. 2,3-BPG binds to a
pocket in the T-state of hemoglobin and is released as it forms the R-state. The presence of 2,3-BPG means that
more oxygen must be bound to the hemoglobin before the transition to the R-form is possible.

Other regulation such as the Bohr effect in hemoglobin can also be depicted via an oxygen-binding curve. By
analyzing the oxygen-binding curve, one can observe that there is a proportional relationship between affinity of
oxygen and pH level. As the pH level decreases, the affinity of oxygen in hemoglobin also decreases. Thus, as
hemoglobin approaches a region of low pH, more oxygen is released. The chemical basis for this Bohr effect is due
to the formation of two salt bridges of the quaternary structure. One of the salt bridges is formed by the interaction
between Beta Histidine 146 (the carboxylate terminal group) and Alpha Lysine 40. This connection will help to
orient the histidine residue to also interact in another salt bridge formation with the negatively charged aspartate 94.
The second bridge is form with the aid of an additional proton on the histidine residue.

As carbon dioxide diffuses into red blood cells, it reacts with water inside to form carbonic acid, which drops the pH
and stabilizes the T state.
An oxygen-binding curve can also show the effect of carbon dioxide presence in hemoglobin. The regulation effect
by carbon dioxide is similar to Bohr effect. A comparison of the effect of the absence and presence of carbon
dioxide in hemoglobin revealed that hemoglobin is more efficient at transporting oxygen from tissues to lungs when
carbon dioxide is present. The reason for this efficiency is that carbon dioxide also decreases the affinity of
hemoglobin for oxygen. The addition of carbon dioxide forces the pH to drop, which then causes the affinity of
hemoglobin to oxygen to decrease. This is extremely evident in the tissues, where the carbon dioxide stored in the
tissues are released into the blood stream, then undergoes a reaction that releases H+ into the blood stream,
increasing acidity and dropping the pH level.
role of hindered environment on the ability of hemoglobin to bind carbon monoxide
-. Your red blood cells (rbc's) are basically little bags of a substance called hemoglobin. In fact, rbc's are
the only cells in your body without nuclei; they lose them when they mature so that more hemoglobin can
be packed into the cell. Hemoglobin molecules are little oxygen carriers. Each molecule has four "seats"
that can be filled with oxygen. They can pick up oxygen in your lungs and deliver it all over your body.
Every cell needs a supply of oxygen. Carbon monoxide (CO) is very dangerous because it sticks to your
hemoglobin better than oxygen does. It "hogs the seats" so that oxygen can't get a ride. And those CO
molecules keep riding around, never giving their seats up to the oxygen. This means there's no way to get
oxygen to your brain, heart, or other cells and those cells start to die. The chemical reactions that stop
happening when there's no oxygen are the ones that make ATP, the form of energy that all of our cells
use.

define p50 and indicate its significant in oxygen transport and delivery

- The P50 is the oxygen tension at which hemoglobin is 50% saturated. The normal P50 is 26.7 mm Hg.

- p50 is a shorthand representation of hemoglobin- oxygen affinity. A lower p50 is protective in ambient
hypoxemia, whereas increasing the p50 should be beneficial in hypoxia due to lung disease, anemia, and
tissue ischemia. ... It is unusual for hemoglobin- oxygen affinity to be considered at these times.

Role of 2,3 BPG in oxygen binding and delivery

- 2,3-bisphosphoglycerate (BPG), also known as 2,3-DPG, promotes hemoglobin transition from a high-oxygen-
affinity state to a low-oxygen-affinity state.2,3-BPG binds to the central compartment of the hemoglobin tetramer
changing its conformation and shifting the oxygen disassociation curve to the right.

Describe the structure of proteins. Proteins are polymers of amino acids, formed by long chains of
amino acids joined together by peptide bonds. ... Secondary structure is the folding of the polypeptide
chain due to hydrogen bonding into an alpha helix structure, resembling a coiled spring, or a beta pleated
sheet.