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APPENDICES
I.
THEORY
PRACTICAL LESSON 1
SIMPLE PROTEINS. STRUCTURE AND FUNCTION
Appendix 1a. Classification and structure of amino acids
More than 300 different amino acids have been described in nature. However,
only 20 of them are commonly found as constituents of mammalian proteins. [Note:
These are the only amino acids that are coded by DNA, the genetic material in the
cell.] Each amino acid (except of proline) has a carboxyl group, an amino group,
and a distinctive side chain or radical ("R-group") bonded to the α-carbon atom
(Figure 1.1 A).
At physiologic pH (approximately pH = 7.4), the carboxyl group is
dissociated, forming the negatively charged carboxylate ion (-COO-), while the
amino group is protonated (-NH3+). In proteins almost all of these carboxyl and
amino groups are combined in peptide chain and, in general, are not available for
chemical reaction except for hydrogen bond formation (Figure 1.1B). Thus, the
nature of the side chains ultimately dictates the role an amino acid in a protein. It is,
therefore, useful to classify the amino acids according to the properties of their side
chains into: nonpolar (have an even distribution of electrons) or polar (have an
uneven distribution of electrons, such as acids and bases).
A. Amino acids with nonpolar side chains
Each of these amino acids has a nonpolar side chain that does not bind or give
off protons or participate in hydrogen or ionic bonds (see Figure 1.2). The side chains
of these amino acids possess lipid-like properties and that promote hydrophobic
interactions.
1. Location of nonpolar amino acids in proteins: In proteins being present in
aqueous solutions, the side chains of the nonpolar amino acids tend to cluster
together in the interior part of the protein molecule (Figure 1.4). This is due to the
hydrophobicity of the nonpolar R-groups which act much like oil droplets that
coalesce in water. The nonpolar side chain R-groups fill up the interior part of the
protein molecule and support its three-dimensional form. At the same time, in
proteins that are located in a hydrophobic environment, like membranes, the
nonpolar side chain groups are located on the surface of the protein, interacting with
the external lipid environment (see Figure 1.4).
2. Proline: The side chain of proline and its α-amino group form a ring
structure. Thus proline differs from other α-amino acids containing an imino-group,
instead of an amino group (Figure 1.5). The unique geometry of proline’s molecule
contributes to the formation of the fibrous structure of collagen, and often interrupts
the α-helices found in globular proteins.
B. Amino acids with uncharged polar side chains
These amino acids have neutral net charge at neutral pH although the side
chains of cysteine and tyrosine can lose a proton at alkaline pH (see Figure 1.3).
Serine, threonine, and tyrosine contain polar hydroxyl groups that can take part in
hydrogen bond formation (Figure 1.6). The side chains of asparagine and
glutamine, contain carbonyl and amide groups, that can also participate in hydrogen
bonds.
1. Disulfide (-S-S-) bond: The side radical of α-amino acid cysteine contains a
sulfhydryl group (-SH), being an important component of the active center of a
great number of proteins. The -SH groups of two cysteines may oxidise with
subsequent conjugation forming a dimer named cystine, which contains a covalent
cross-link called a disulfide bond (-S-S-).
2. Side chain radicals as sites of reaction with other compounds: A number of αamino acids like Serine, threonine, and tyrosine contain a polar hydroxyl group (OH) that serves as a site of attachment for reactive groups such as inorganic
phosphate group. Moreover, asparagine’s amide group, as well as hydroxyl group
of serine or threonine, can act as a site of binding for oligosaccharide chains of
glycoproteins.
C. Amino acids with acidic side chains
Two α-amino acids, aspartic and glutamic acids, are proton donors. At
neutral pH the side chain radicals of these amino acids are fully ionized, containing
a negatively charged carboxyl group (-COO-). Consequently, these amino acids are
called aspartate or glutamate in order to indicate that these amino acids are
negatively charged at physiologic pH (see Figure 1.3).
D. Amino acids with basic side chains
The side chain radicals of basic amino acids accept protons (see Figure 1.3).
At physiologic pH the side radicals in lysine and arginine residues are positively
charged. In contrast, histidine is slightly basic. Therefore, it is mainly uncharged at
physiologic pH being a free amino acid. However, when histidine is incorporated
into a polypeptide chain, its side radical can be either positively charged or neutral,
depending on the ionic environment provided by the polypeptide chains of the
protein. This important feature of histidine contributes to its role in the proteins’
function (e.g. hemoglobin).
Abbreviations and symbols for the commonly occurring amino acids
Each amino acid name has an associated three-letter abbreviation and a oneletter symbol (Figure 1.7). The one-letter codes are determined by the following
rules:
1. Unique first letter: If only one amino acid begins with a particular letter, then
that letter is used as its symbol. For example, I = isoleucine.
2. Most commonly occurring amino acids have priority: If a number of one amino
acids begins with a similar letter, the most prevalent amino acid receives this letter
as its symbol. For example, threonine is more prevalent in comparison to Tyrosine,
so T is a symbol of threonine.
3. Similar sounding names: Someone -letter symbols remind the amino acid they
represent by sound (e.g., F = phenylalanine (ph = [f]) or W= tryptophan
("tWyptophan" by Elmer Fudd).
4. Letter close to initial letter: In the case of remaining amino acids, their oneletter symbol is designated as close in the alphabet as possible to the initial letter of
the acid. For example, the letter “B” designates Asx (aspartic acid or asparagine)
being located near “A” in the alphabet.
F. Optical properties of amino acids
The α-carbon atom of each amino acid (except of glycine) is binded to four
different chemical groups and, consequently, is optically active (chiral) carbon atom.
The α-carbon atom in the glycine’s molecule has two hydrogen substituents (3
different groups instead of 4 for other amino acids). Thus, glycine is optically
inactive. Amino acids with an asymmetric center at the α-carbon exist in two
different optical forms, designated as D- and L-isomers, being mirror images of each
other (Figure 1.8). These two forms are termed as stereoisomers, optical isomers,
or enantiomers. All amino acids being present in mammalian proteins are of the Lisomers. At the same time, D-amino acids are present in bacterial cell walls proteins.
Appendix 1b. Acid-Base Behavior of Amino Acids
Every α-amino acid contains both basic amino (NH2) and acidic carboxyl
(COOH) groups. Consequently, hydrogen (proton) transfer from acidic to the basic
group leads to “zwitterion” formation (from German, where “zwitter” means
“double”). This zwitterion is a salt containing both single positive and single
negative charges at distinct functional groups. Taking into account the presence of
both positive (+1) and negative (-1) charges in the zwitterion, its net charge is neutral
(0).
Actually, an amino acid exists in different forms, depending on the reaction
of the current medium. When the reaction of the medium is near-neutral (pH ~ 6),
alanine and other neutral amino acids exist in zwitterionic forms (A) without net
charge (0). In zwitterionic form the carboxyl group is negatively charged being a
carboxylate anion- (-COO-) whereas the amino group is positively charged being an
ammonium cation (-NH3+).
When the reaction of the medium becomes acidic (pH = 2 or even lower), the
carboxylate anion binds a proton originating from the acidified media becoming
neutral (carboxyl group). At the same time, amino acid has a net positive (+1) charge
(form B).
When the reaction of the medium becomes alkaline (pH > 10) the ammonium
cation loses a proton (the latter reacts with hydroxyl groups originating from the
medium forming a molecule of water) and the amino acid has a net negative (-1)
charge (form C).
Thus, alanine exists in one of three different forms depending on the pH of the
medium solution. At the physiological pH of 7.4, neutral amino acids are present
mainly in zwitterionic forms.
The value of pH at which the amino acid exists in zwitterion (neutral charge)
is called its isoelectric point, abbreviated as pI.
The pI of neutral amino acids are generally equal to pH ~ 6. Acidic amino acids,
having one more carboxyl group, have lower pI values (around 3). The three basic
amino acids, which have one more amine group that is able to accept a proton, have
higher pI values (around 7.6-10.8).
Appendix Ic. Peptide Bond Formation
During formation of a dipeptide, the amine (–NH2) group of one amino acid
forms an amide bond with the carboxyl (-COOH) group of another amino acid. A
proton (H+) originating from amine group and hydroxyl anion (HO-) from carboxyl
group react leading to the formation of water (H2O) that is released into the medium.
For example, reaction of the -COO- group of alanine with the –NH3+ group of serine
forms a dipeptide with one new amide bond. The dipeptide has an ammonium cation
(–NH3+) at one end of its chain and a carboxylate anion (-COO-) at the other:
When two or more amino acids are joined together by amide bonds, forming large
molecules called peptides and proteins. A dipeptide has two amino acids connected
to each other by one amide bond. A tripeptide has three amino acids joined by two
amide bonds:
Molecules containing from 12 to 20 amino acid residues are called oligopeptides.
The ones containing more than 20 amino acids are polypeptides. Proteins are
macromolecules with molecular weight from 5000 and more consisting of one or
more polypeptide chains.
The amide bonds in peptides and proteins are called peptide bond. Individual
amino acids are called amino acid residues. The names of residues are formed by
replacing the suffix –ine or –ate with –yl. For example, glycine residue in the peptide
chain is called glycyl while glutamate residue is called glutamyl. In the cases of
asparagine, glutamine and cysteine, -yl replaces the final –e forming asparaginyl,
glutaminyl, and cysteinyl, respectively.
Every polypeptide chain has a number of universal structural properties. At
the one end of the chain one free α-amino group is located. This end is called the
amino terminal (N-terminal) end and this amino acid is named as the first amino
acid. The other end of the polypeptide chain is the carboxy-terminal end (Cterminal), where a free α-carboxyl group which is contributed by the last amino
acid is located.
Appendix Id. Protein folding.
Role of chaperones in protein folding
New polypeptides are synthesized de novo in the cell by a translation complex
including ribosomes, mRNA, and various factors. As the newly synthesized
polypeptide chain is released from the ribosome, it folds into its three-dimensional
shape. Folded proteins take up a low-energy state that makes the native structure
more stable. In most cases the native conformation is reached in less than a second,
indicating that folding is a very rapid process.
Protein folding and stabilization depend on disulfide bonds and several
noncovalent forces including the hydrophobic effect, hydrogen bonding, van der
Waals interactions, and charge-charge interactions. The weakness of each
noncovalent interactions allow proteins to undergo small conformational changes.
This results in a correctly folded protein with a low energy state. When new proteins
are not folded correctly, they may react with other proteins and consequently form
aggregates. A number of neurodegenerative disorders, such as Alzheimer’s,
Parkinson’s, Huntington’s diseases, are caused by accumulation of protein deposits
from such aggregates.
Due to the high rapidity of protein folding this process should be determined
by the primary structure of the polypeptide chain.
The outcome of correctly folded proteins is up-regulated by a group of
specialized proteins, molecular chaperones. Originally, the term “chaperone” was
used for designation of an older person who accompanies the younger one(s) to
ensure the right behavior. These proteins bind to the polypeptides before their
folding
is complete. It allows to prevent the formation of additional inter- and intramolecular
bonds leading to incorrect folding and impaired protein structure. Moreover,
chaperones may bind unassembled protein subunits preventing their conjugation
before they are combined into a complete complex protein.
A wide number of chaperones present in the living cell. However, most of
them are heat shock proteins (HSPs) - proteins that are synthesized in response to an
increase of temperature (heat shock) or other changes that cause protein denaturation
in vivo. The role of HSPs is to repair the damage caused by denaturating factors by
binding to denatured proteins and helping them to refold into their native
conformation.
Appendix Ie. Classification of proteins
Peptide Classification
Peptide is the term indicating short polymers of amino acids. Peptides are
classified by the number of amino acids in the polypeptide chain. Each amino acid
in the chain is called an amino acid residue, indicating the fragment left after the
release of water resulting from the formation of peptide bond (Appendix Ic).
Dipeptides have two amino acid residues, tripeptides – 3, tetrapeptides – 4, and
so on. However, when the number of amino acid residues in the chain exceeds 12
but not 20 these polypeptides are called oligopeptides. If the peptide contains more
than 20 residues, it is called polypeptide.
Proteins Are Composed of One or More Polypeptide Chains
For indication of single polypeptide chains both polypeptide and protein can
be used. However, the term protein more often indicates a molecule composed of
one or more polypeptide chains. The terms polypeptide and protein are used
interchangeably in discussing single polypeptide chains. If the protein molecule has
only one polypeptide chain, this protein is called monomeric protein. When there
is more than one chain the protein molecule, it is called multimeric (di-; trimeric
etc.) protein. If the structure of polypeptides in the protein is equal, this protein is
called homomultimeric. Oppositely, when the structure of polypeptides in the
protein molecule is distinct, this protein is heteromultimeric. Greek letters and
subscripts describe the peptide composition of multimeric proteins. Thus, an α2-type
protein is a dimer of identical polypeptide subunits, or a homodimer. Hemoglobin
consists of four polypeptides of two different kinds; it is an α2β2 heteromultimer.
Architecture of Protein Molecules
Protein Shape
All present proteins can be classified into one of three global classes on the
basis of shape and solubility: fibrous, globular, or membrane.
1. Fibrous proteins have relatively simple, regular linear structures. These proteins
carry out structural function in cells. Commonly, they are not soluble in aqueous
solutions.
2. Globular proteins have spherical shape. They are folded so that hydrophobic
amino acid side chains are located in the interior of the molecule in order to avoid
contact with water. Hydrophilic side amino acid residues are located on the surface
of the molecule contacting with water. Consequently, globular proteins are
characterized by good solubility in water. Cytosolic enzymes being soluble are
globular in shape.
3. Membrane proteins are structurally associated with biological membranes.
Hydrophobic amino acid residues are located inside the membrane for interaction
with nonpolar phase presented by lipids. In this connection, membrane proteins are
insoluble in aqueous solutions. However, in experimental procedures these proteins
may be solubilized with various detergents. Membrane proteins in comparison with
cytosolic proteins have less hydrophilic amino acids. The latter are located outside
the membrane (Figure 1.4; Practical lesson I).
Biological Functions of Proteins
Proteins are biologically active agents. Proteins take part in all metabolic
processes inside the cell. That is why one of the proteins classifications is based on
their biological function.
1. Enzymes
Enzymes represent the widest class of proteins (more than 3000 individual
enzymes in the Enzyme Nomenclature and Enzyme Classification).
Enzymes act as biological catalysts that accelerate the rates reactions inside
the living organism. Each Enzymes are specific in their action and modulate
individual metabolic reactions (Practical lesson 3). Virtually every step of the
metabolism is catalyzed by one or more enzymes. The catalytic activity of enzymes
manifold exceeds the one observed for inorganic catalysts. Enzymes enhance the
reaction rates to maximum of 1016-fold in comparison to the spontaneous
uncatalyzed reaction. Enzymes are classified and named according to the type of the
reaction they catalyze (glutathione transferase (GST) transfers glutathione into the
acceptor molecule, whereas glutathione reductase (GR) catalyses reduction of
oxidized glutathione into reduced glutathione). The systematic names of enzymes
come from the type and the participants of the reaction they catalyze. For example,
the name “glyceraldehyde-3-phosphate:NAD oxidoreductase” means that the
substrate of the reaction is glyceraldehyde-3-phosphate, the reaction type is
reduction/oxidation reaction in presence of the cofactor NAD. At the same time, the
enzymes have also common names being less massive. For example, the above
mentioned enzyme is also known as “glyceraldehyde-3-phosphate dehydrogenase”.
A number of enzymes have trivial names with historical origin. For example, an
antoxidant enzyme catalyzing decomposition of hydrogen peroxide catalase is
systematically called as hydrogen-peroxide:hydrogen-peroxide oxidoreductase.
2. Regulatory Proteins
A number of proteins do not catalyze obvious chemical reactions, however,
they regulate the ability of other proteins to carry out their physiological functions.
These proteins are called regulatory proteins. For example, insulin (from latin
“insula” – an islet), a pancreatic hormone being a protein (5.7 kD), consists of two
polypeptide chains (21 and 30 amino acid residues) connected to each other by
disulfide bods (cross-bridges).
Other proteins take part in regulation of gene expression. They bind to certain
DNA sites activating or inhibiting the transcription of genetic information from
DNA to RNA. For example, repressor proteins (or repressors) block the
transcription process being negative regulators of transcription. Positive peptide
regulators of transcription also present in the living cell.
3.Transport Proteins
Transport proteins represent the third functional class of proteins. These
proteins transport a wide number of chemical substances from one site of the cell or
an organism to another. The most obvious example is hemoglobin that transfers
oxygen from lungs to other tissues. Another type of transport is accompanied by the
transport of different substances across biological membranes mediated by specific
proteins. Membrane transport proteins (or in some cases just “membrane
transporters”) transfer chemical compounds from the one side of the membrane to
another. For example, family of glucose transporter (GluT) proteins transfer glucose
from blood into the cell through cellular membrane.
4.Storage Proteins
A number of proteins provide reservoirs for different chemicals (most often the
latter are essential) being storage proteins. Obviously, proteins are reservoirs of
nitrogen (e.g. ovalbumin or egg albumin). At the same time, other proteins store
essential trace elements like iron in the case of ferritin. The latter is able to bind and
store up to 4500 iron atoms.
5.Contractile Proteins
A number of proteins allow cells to move in the medium.
The contractile and motile proteins have a number of common properties. These
proteins are filamentous or polymerize to form filaments. For example, actin and
myosin interact forming contractile systems of the cell. Motor proteins like kinesis
drive the movement of certain organelles.
6.Structural Proteins
An important role of proteins consists in formation of the biological structures.
These proteins also polymerize generating long and extra-long fibers. For example,
collagen is an insoluble fibrous protein being a structural part of connective tissues,
bones, forming dense extra-strong fibrils. Oppositely, elastin is a protein with elastic
properties. It is an important component of elastic connective tissues.
7.Scaffold Proteins (Adapter Proteins)
Scaffold or adapter proteins proteins play a significant role in the complex
pathways of cellular response to hormones and growth factors. Adaptor proteins
contain a large variety of protein-binding modules that link protein-binding
elements together and facilitate the creation of larger signaling complexes.
Generally, these proteins enhance protein-protein interactions leading to the
formation of protein complexes.
8.Anchoring (targeting) proteins bind other proteins leading to their
association with other cellular structures. , causing them to associate with other
structures in the cell. Particularly, a family of anchoring proteins, known as AKAP
or A kinase anchoring proteins, exists in which specific AKAP members bind the
regulatory enzyme protein kinase A (PKA) to particular subcellular compartments.
9.Protective and Exploitive Proteins
A number of proteins are protective and exploitive because of their role in
defense and protection. The most expressed defensive action is characteristic for
immunoglobulins that recognize and neutralize foreign bacterial, viral and other
antigens without affecting cells and tissues of the host organism.
Simple proteins and conjugated proteins
Many proteins consist of polypeptide chain(s) and contain no other chemical
groups. Such proteins are called simple proteins. At the same time, many other
proteins contain various chemical constituents as an integral part of their structure.
These proteins are termed conjugated proteins. If the nonprotein part is strongly
binded to the protein’s molecule even by covalent bonds, it is called a prosthetic
group. If the nonprotein component is not covalently linked to the protein molecule,
it is called coenzyme. Сonjugated proteins are classified according to the chemical
nature of nonprotein part.
1.GLYCOPROTEINS. Glycoproteins are proteins that contain a
carbohydrate fragment in their structure. Proteins located extracellularly are often
glycoproteins. For example, proteoglycans are important components of the
extracellular matrix. Moreover, many transmembrane proteins are glycosylated on
their extracellular sites.
2. LIPOPROTEINS. Lipoproteins are protein molecules containing lipid
component in the structure. The most representative examples of this class of
proteins are blood plasma lipoproteins. These lipoproteins act as lipid transporters
from the site of synthesis to target cells and tissues. The ratio between different
serum lipoprotein particles especially low-density lipoproteins (LDLs) and highdensity lipoproteins (HDLs) indicate the risk of atherosclerosis.
3. NUCLEOPROTEINS. Protein and nucleic acid conjugates termed
nucleoproteins have many roles in the storage and transmission of genetic
information.
4. PHOSPHOPROTEINS. Phosphoproteins are characterized by the
presence of phosphate groups binded to hydroxyls of serine, threonine, or tyrosine
residues. Regulation of protein active or inactive state is realized through
phosphorylation (connection of phosphate group) of its center.
5. METALLOPROTEINS. Metalloproteins are proteins that contain metal
ions (most often transition metal ion) in their active center. In the case of enzymes,
these enzymes are called metalloenzymes. At the same time, metal-storage proteins
like ferritin also may be classified as metalloproteins.
6. HEMOPROTEINS. Hemoproteins contain heme as a prosthetic group in
their structure. The most obvious example of this class is hemoglobin.
7. FLAVOPROTEINS. Flavoproteins are chromoproteins (“coloured
proteins”) containing the derivatives of flavin, FMN and FAD, as prosthetic groups.
Appendix If.
PRACTICAL LESSON 5
WATER-SOLUBLE AND LIPID- SOLUBLE VITAMINS. VITAMINS AS
COFACTORS
Nomenclature and classification of vitamins
Vitamins are chemically unrelated organic compounds that being essential
cannot be synthesized by humans and, therefore, must be supplied by the diet. Nine
vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin,
riboflavin, biotin, and pantothenic acid) are classified as water-soluble, whereas four
vitamins (vitamins A, D, E and K) are termed lipid-soluble. Vitamins are required
for specific cellular functions. For example, many of the water-soluble vitamins are
precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to
the water-soluble vitamins, only one fat soluble vitamin (vitamin K) has a coenzyme
function. These vitamins are released, absorbed and transported with dietary lipids.
They are not readily excreted in the urine, and significant quantities are stored in the
liver and adipose tissue. In fact, consumption of vitamins A and D in excess of the
recommended dietary allowances can lead to accumulation of toxic quantities of
these compounds. A vitamin-deficient state (disease) can occur when a vitamin is
deficient or absent in the diet. Such diseases can be treated or prevented by
consumption of the respective vitamin.
Classification of the Vitamins