Download 1.0 amino acids as units of protein structure

1.0 AMINO ACIDS AS UNITS OF PROTEIN STRUCTURE
The covalent backbone of a typical protein contains hundreds of individual bonds. Because free
rotation is possible around many of these bonds, the protein can assume an unlimited number of
conformations. However, each protein has a specific chemical or structural function, strongly
suggesting that each has a unique three-dimensional structure. The spatial arrangement of atoms
in a protein is called its conformation. The possible conformations of a protein include any
structural state that can be achieved without breaking covalent bonds. A change in conformation
could occur, for example, by rotation about single bond. Proteins in any of their functional, folded
conformations are called native proteins.
1.1
Peptide Linkage and Peptides.
The –COOH group of one amino acid can be joined to the –NH2 group of another by a covalent
bond called peptide bond. In the process of formation of a peptide bond, a molecule of water is
eliminated.
Figure 1: A peptide bonds formation between two amino acids
When two amino acids are joined together by one peptide bond, such a structure is called a
dipeptide. A third amino acid can form a second peptide bond through its free –COOH end and is
called tripeptide. Thus, it is the peptide linkage which holds various amino acids together in a
specific sequence and number. Peptide varying from the simplest dipeptide to very long
polypeptides are present in human body and serve specific functions. Peptide chains that contain
fewer than 50 amino acid residues are called peptides or oligopeptides.
1.2 Characteristics of a peptide bond
2. The C-N backbones in each peptide group have some double-bond character and do not
rotate.
3. The other backbone bonds designated Cα-N and Cα-C, are theoretically free to rotate, since
they are true single bonds.
4. But if the R group attached to the central α carbon alone is large enough, it will prevent
complete rotation around the Cα-N and Cα-C.
Figure 2: A free rotation formed between Cα-N and Cα-C bonds.
1.3 Structural Organisation of Proteins
Protein structure is normally described at four levels of organisation; Primary, Secondary, Tertiary
and Quaternary structures.
1.31 Primary Structure
Primary structure is the linear sequence of amino acids held together by peptide bonds in its peptide
chain. The free –NH2 group of the terminal amino acid is called as N-terminal end and the free –
COOH end is called as C-terminal end. It is a tradition to number the amino acids from N-terminal
ends as No. 1 towards the C-terminal end. Presence of specific amino acids at a specific number
is very significant for a particular function of a protein. Any change in the sequence is abnormal
and may affect the function and properties of protein.
1..32 Secondary Structure
The polypeptide backbone does not assume a random three-dimensional structure, but instead
generally forms regular arrangements of amino acids that are located near to each other in the
linear sequence. These arrangements are termed the secondary structure of the polypeptide. The
linkage or bonds involved in the secondary structure formation are hydrogen bonds and
disulphide bonds. Hydrogen bonds are weak, low energy covalent bonds sharing a single
hydrogen by two electronegative atoms such as O and N. Hydrogen bonds are formed in secondary
structure by sharing H-atoms between oxygen of CO and –NH of different peptide bonds. The
hydrogen bonds may from either α-helix or β-pleated sheet structure. Disulphide bonds are formed
between two cysteine residues. They are strong, high energy covalent bonds.
α-helix
Several different polypeptide helices are found in nature, but the α-helix is the most common. It is
a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side
chains of the component amino acids extending outward from the central axis to avoid interfering
sterically with each other. The coils are stabilized by hydrogen bonds between carbonyl O of 1st
amino and amide N of 4th amino acid residue. Thus, in α-helix intra chain hydrogen bonding is
present.
Each amino acid residue advances by 0.15 nm long the helix, and 3.6 amino acid residues are
present in one complete turn. The distance between two equivalent point on turn is 0.54nm and is
called a pitch. Small or uncharged amino acid residues such as alanine, leucine and phenylalanine
are often found in α-helix. The protein of hair, nail, skin contain a group proteins called keratins
rich in α-helical structure.
Figure 3: A typical hydrogen bond formed between an intra amino acid chain to form an α-helical structure
β-pleated sheet structure
The β -sheet is another form of secondary structure in which all of the peptide bond components
are involved in hydrogen bonding. The surfaces of β-sheets appear “pleated,” and these
structures are, therefore, often called β -pleated sheets. β-keratins present in spider’s web, reptilian
claw, fibres of skin form almost fully extended chain. The conformation β-pleated sheet structure
is thus formed when hydrogen bonds formed between the carbonyl oxygens and amide hydrogens
of two or more adjacent polypeptide chains. Thus the hydrogen bonding in β-pleated sheet
structure in interchain. The adjacent chains in β-pleated sheet structure are either parallel or
antiparallel depending either the peptide linkage of the chains runs in the same or opposite
direction.
Figure 4: A typical β -pleated sheets structure of amino acid showing parallel and anti-parallel organisation.
Other organisation formed in secondary structure of protein include; β-bends, β-meander, β-α-β
unit, Greek key.
1.33 Tertiary structure
The polypeptide chain with secondary structure may be further folded, superfolded twisted about
itself forming many sizes. Such structural conformation is called tertiary structure. It is only one
conformation which is biologically active and protein in this conformation is called a native
protein. Thus, the tertiary structure is constituted by steric relationship between the amino acids
located far apart but bright together by folding. The bonds responsible for interaction between
groups of amino acids are as follows;
Hydrophobic interactions: normally occur between nonpolar side chains of amino acids such as
alanine, leucine, methionine, isoleucine and phenylalanine. The constitute the major stabilizing
forces for tertiary structure.
Hydrogen bonds: Normally formed by the polar side chains of the amino acids.
Ionic of electrostatic interactions: These are formed between oppositely charged polar side
chains of amino acids, such as basic and acidic amino acids.
Van der Waal forces: occur between nonpolar side chains.
Disulphide bonds: These are S-S bonds between –SH groups of distant cysteine residues.
1.34 Quaternary Structure
Many proteins are made up of only one peptide chain. However, when a protein consists of two or
more peptide chains held together by non-covalent interactions or by cross-links, it is referred to
as quaternary structure. The assembly is often called as oligomer and each constituent peptide
chain is called as monomer or subunit. The monomers of oligomeric protein can be identical or
quite different in primary, secondary and quaternary structure. Haemoglobin and lactate
dehydrogenase
are
tetramers
consisting
of
transcarbamoylase has 72 subunits in its structure.
four
monomers.
An
enzyme
aspartate
2.0
GLYCOLYSIS AS ENERGY METABOLIC PATHWAY
In a pathway, the product of one reaction serves as the substrate of the subsequent reaction.
Different pathways can also intersect, forming an integrated and purposeful network of chemical
reactions. These are collectively called metabolism, which is the sum of all the chemical
changes occurring in a cell, a tissue, or the body. Most pathways can be classified as either
catabolic (degradative) or anabolic (synthetic). Catabolic reactions break down complex
molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules, for example,
CO2, NH3 (ammonia), and water. Anabolic pathways form complex end products from simple
precursors, for example, the synthesis of the polysaccharide, glycogen, from glucose.
In the glycolysis pathway, a molecule of glucose is converted in 10 enzyme catalyzed steps to two
molecules of 3-carbon pyruvate. Hence, glycolysis is defined as the oxidation of glucose or
glycogen to pyruvate and lactate with the aim to produce energy in form of ATP molecules to the
cells. It virtually occurs in all tissue. Red blood cells and nervous tissues derive its energy mainly
from glycolysis. This pathway is unique in the sense that it can utilize O2 if available (aerobic)
which produces more ATP and it can function in the absence of O2 also (anaerobic). This process
of glycolysis occurs in the cytosol of the cell.
In Aerobic condition, reduced NAD in form of NADH + H+ in the presence of O2 is oxidized in
electron transport chain (ETC) producing ATP. While in Anaerobic condition, NADH + H+
cannot be oxidised in ETC, so no ATP is produced in ETC. But NADH + H+ is oxidised to NAD+
by conversion of pyruvate to lactate without producing ATP.
Why is glycolysis so important to organisms?
For some tissues (such as brain, kidney medulla, and rapidly contracting skeletal muscles) and
for some cells (such as erythrocytes and sperm cells), glucose is the only source of metabolic
energy. In addition, the product of glycolysis—pyruvate—is a versatile metabolite that can be used
in several ways.
2.1 Stages involved in Glycolysis to produce ATP are as follows;
Two stages are involved in the breakdown of glucose; The preparatory stage which consumes ATP
and the payoff stage which produces ATP.
The Preparatory stage;
1.
Phosphorylation of Glucose in the first step of glycolysis, glucose is activated for subsequent
reactions by its phosphorylation at C-6 to yield glucose 6-phosphate, with ATP as the
phosphoryl donor. This reaction, which is irreversible under intracellular conditions, is
catalyzed by hexokinase, which requires Mg2+ as a cofactor.
2.
Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate The enzyme phosphohexose
isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6
phosphate, an aldose, to fructose6-phosphate, a ketose
3.
Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate in the second of the
two priming reactions of glycolysis, phosphofructokinase-1 (PFK-1) catalyzes the transfer
of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate:
The PFK-1 reaction is essentially irreversible under cellular conditions
4.
Cleavage of Fructose 1,6-Bisphosphate The enzyme fructose 1,6-bisphosphate aldolase,
often called simply aldolase, catalyzes a reversible aldol condensation. Fructose 1,6
bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde
3-phosphate, an aldose, and dihydroxyacetone phosphate, a ketose:
5.
Interconversion of the Triose Phosphates Only one of the two triose phosphates formed by
aldolase, glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of
glycolysis. The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted
to glyceraldehyde 3-phosphate by the fifth enzyme of the sequence, triose phosphate
isomerase.
Payoff Stage;
6.
Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate The first step in the
payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate,
catalyzed by glyceraldehyde 3- phosphate dehydrogenase.
7.
Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP The enzyme phosphoglycerate
kinase
transfers
the
high-energy
phosphoryl
group
from
the
carboxyl
group
of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate
8.
Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate The enzyme phosphoglycerate
mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate;
Mg2+ is essential for this reaction
9.
Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate In the second glycolytic reaction
that generates a compound with high phosphoryl group transfer potential, enolase promotes
reversible removal of a molecule of water from 2-phosphoglycerate to yield
phosphoenolpyruvate (PEP)
10.
Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP. The last step in
glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to
ADP, catalyzed by pyruvate kinase, which requires K+ and either Mg2+ or Mn2+
Glycolysis consists of two phases. In the first phase, a series of five reactions, glucose is broken
down to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent
reactions convert these two molecules of glyceraldehyde-3-phosphate into two molecules of
pyruvate. Phase 1 consumes 2 molecules of ATP. The later stages of glycolysis result in the
production of 4 molecules of ATP. The net is 4-2=2 molecules of ATP produced per molecule of
glucose breakdown in glycolysis. In aerobic condition, the 2 molecules of NADH + H+ produced
are utilized in a chain of reaction that occurs in the mitochondria called an Electron Transport
Chain (ETC), to produce 3 molecules of ATP per NADH + H+ molecule. Hence, 6 molecules of
ATP are produced from the use of NADH + H+ in ETC. Therefore, a total of 8 molecules of ATP
is produced through glycolysis in the cells in Aerobic condition. Glycolysis is also called
Embden-Meyerhof pathway. While in an Anaerobic condition, 2molecules of ATP are produced
which is from the pathway of glycolysis. NADH + H+ cannot be oxidised in ETC, so no ATP is
produced in ETC. But NADH + H+ is oxidised to NAD+ by conversion of pyruvate to lactate
without producing ATP.
Figure 5: Glycolytic pathway
3.0
THE ISOLATION AND PURIFICATION OF PROTEIN
Cells contain thousands of different proteins. A major problem for protein chemists is to purify a
chosen protein so that they can study its specific properties in the absence of other proteins.
Because the biological function of a protein depends on its native structure, techniques employed
in protein purification should not denature the protein. The starting material for protein purification
is usually a cell type or tissue of interest; alternatively, cloned cells engineered to mass produce
the protein are used.
Proteins can be separated and purified on the basis of their two prominent physical properties: size
and electrical charge. A more direct approach is to use affinity purification strategies that take
advantage of the biological function or specific recognition properties of a protein. Separation
methods based on size include size exclusion chromatography, ultrafiltration, and
ultracentrifugation are implemented. The ionic properties of peptides and proteins are determined
principally by their complement of amino acid side chains. Furthermore, the ionization of
these groups is pH dependent. A variety of procedures has been designed to exploit the electrical
charges on a protein as a means to separate proteins in a mixture. These procedures include ion
exchange chromatography, electrophoresis. Using solubility properties to separate, proteins tend
to be least soluble at their isoelectric point, the pH value at which the sum of their positive and
negative electrical charges is zero. At this pH, electrostatic repulsion between protein molecules
is minimal and they are more likely to coalesce and precipitate out of solution. Ionic
strength also profoundly influences protein solubility. Most globular proteins tend to become
increasingly soluble as the ionic strength is raised. This phenomenon, the salting-in of proteins, is
attributed to the diminishment of electrostatic attractions between protein molecules by the
presence of abundant salt ions. Such electrostatic interactions between the protein molecules would
otherwise lead to precipitation. Although the side chains of nonpolar amino acids in soluble
proteins are usually buried in the interior of the protein away from contact with the aqueous
solvent, a portion of them may be exposed at the protein’s surface, giving it a partially hydrophobic
character. Hydrophobic interaction chromatography is a protein purification technique
that exploits this hydrophobicity.
Table 1: Example of a Protein Purification Scheme: Purification of an Enzyme from a Cell Extract
Dialysis
If a solution of protein is separated from a bathing solution by a semipermeable membrane, small
molecules and ions can pass through the semipermeable membrane to equilibrate between the
protein solution and the bathing solution, called the dialysis bath or dialysate. This method is
useful for removing small molecules from macromolecular solutions or for altering the
composition of the protein-containing solution.
Ultrafiltration
This is an improvement on the dialysis principle. Filters with pore sizes over the range of
biomolecular dimensions are used to filter solutions to select for molecules in a particular size
range.
Because
the
pore
sizes
in
these
filters
pressures are often required to force the solution through the filter.
are
microscopic,
high
Ion Exchange Chromatography
Charged molecules can be separated using ion exchange chromatography, a process in which the
charged molecules of interest (ions) are exchanged for another ion (usually a salt ion) on a charged
solid support. In a typical procedure, solutes in a liquid phase, usually water, are passed through a
column filled with a porous solid phase composed of synthetic resin particles containing charged
groups. Resins containing positively charged groups attract negatively charged solutes and are
referred to as anion exchange resins. Resins with negatively charged groups are cation exchangers.
Increasing the salt concentration in the solution passing through the column leads to competition
between the cationic proteins bound to the column and the cations in the salt for binding to the
column. Bound cationic proteins that interact weakly with the charged groups on the resin wash
out first, and those inter acting strongly are washed out only at high salt concentrations.
Size Exclusion Chromatography
Size exclusion chromatography is also known as gel filtration chromatography or molecular sieve
chromatography. In this method, fine, porous beads are packed into a chromatography column.
The beads are composed of dextran polymers, agarose, or polyacrylamide. The pore sizes of these
beads approximate the dimensions of macromolecules. As a solution of molecules is passed
through the column, the molecules passively distribute between the pores based on their sizes.
Electrophoresis
Electrophoretic techniques are based on the movement of ions in an electrical field. If a mixture
of electrically charged biomolecules is placed in an electric field of field strength E, they flow
freely towards an electrode with an opposite charge. Although, movement by every molecule
differs in relation to the physical characteristics of the molecule and experimental system used. In
an electric field, the velocity of movement, ν, of a charged molecule depends on variables
described by (v=E.q/f) where f is the frictional coefficient (which describes the frictional resistance
to mobility and depends on a number of factors such as mass of the molecule, its degree of
compactness, buffer viscosity and the porosity of the matrix in which the experiment is performed),
q is the net charge on the molecule (determined by the abundance of positive and negative charges
in the molecule such as charges on proteins due to the amino acid side chains). This equation
explains that molecules will move faster as their net charge increases, the electric field strengthens
and as f decreases (which is a function of molecular mass/shape). Molecules of similar net charge
separate due to differences in frictional coefficient while molecules of similar mass/shape may
differ widely from each other in net charge Generally, electrophoresis is carried out not in free
solution but in a porous support matrix such as polyacrylamide or agarose, which retards the
movement of molecules according to their dimensions relative to the size of the pores in the matrix.
Hydrophobic Interaction Chromatography
Hydrophobic interaction chromatography (HIC) exploits the hydrophobic nature of proteins in
purifying them. Proteins are passed over a chromatographic column packed with a support matrix
to which hydrophobic groups are covalently linked. Phenyl Sepharose, an agarose support matrix
to which phenyl groups are attached, is a prime example of such material. In the presence of high
salt concentrations, proteins bind to the phenyl groups by virtue of hydrophobic interactions.
Proteins in a mixture can be differentially eluted from the phenyl groups by lowering the salt
concentration or by adding solvents such as polyethylene glycol to the elution fluid.
High-Performance Liquid Chromatography
The principles exploited in high-performance (or high-pressure) liquid chromatography (HPLC)
are the same as those used in the common chromatographic methods such as ion exchange
chromatography or size exclusion chromatography. Very-high-resolution separations can be
achieved quickly and with high sensitivity in HPLC using automated instrumentation. Reversephase HPLC is a widely used chromatographic procedure for the separation of nonpolar solutes.
In reverse-phase HPLC, a solution of nonpolar solutes is chromatographed on a column having a
nonpolar liquid immobilized on an inert matrix; this nonpolar liquid serves as the stationary phase.
A more polar liquid that serves as the mobile phase is passed over the matrix, and solute molecules
are eluted in proportion to their solubility in this more polar liquid.
Affinity Chromatography
Affinity purification strategies for proteins exploit the biological function of the target protein. In
most instances, proteins carry out their biological activity through binding or complex formation
with specific small biomolecules, or ligands, as in the case of an enzyme binding its substrate. If
this small molecule can be immobilized through covalent attachment to an insoluble matrix, such
as a chromatographic medium like cellulose or polyacrylamide, then the protein of interest, in
displaying affinity for its ligand, becomes bound and immobilized itself. It can then be removed
from contaminating proteins in the mixture by simple means such as filtration and washing the
matrix. Finally, the protein is dissociated or eluted from the matrix by the addition of high
concentrations of the free ligand in solution.