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05/06/2018
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Protein structure and function
The secondary structural elements and their
interactions are the building blocks for the movable
and dynamic elements in proteins! If we want to
understand these motions, we need to know what is
out there and how to describe it
Amino Acids differing only in their side chains compose
proteins
The monomeric building blocks of proteins are 20 amino acids (L isomer), which
have a characteristic structure consisting of a central C bonded to four different
chemical groups: an amino group, a carboxylic acid group, a hydrogen atom, and
one variable group, called a side chain or R group.
Amino acids can be polymerized to form chains. The resulting CO-NH linkage, an
amide linkage, is known as a peptide bond.
N-terminus C-terminus
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Amino acids can be classified into several broad categories based on the size,
shape, charge, hydrophobicity, and chemical reactivity of the side chains. According
to the most common classification scheme, by the polarities of their side chains,
there are three major types of amino acids:
1. those with nonpolar R groups
2. those with uncharged polar R groups, and
3. those with charged polar R groups
1. The nonpolar amino acids side chains have a variety of shapes and sizes. Nine
amino acids are classified as having nonpolar side chains: Gly, Ala, Val, Leu, Ile, Met,
Pro, Phe, Trp.
Ala
Ile
Phe
Amino acids with nonpolar side chains are hydrophobic and so poorly soluble in water.
Absorption of ultraviolet light
by aromatic amino acids.
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Amino acids with polar side chains are hydrophilic; the most hydrophilic of these amino
acids is the subset with side chains that are charged at the pH = 7
2. Uncharged polar side chains have hydroxyl, amide or thiol groups. Six
amino acids are commonly as having uncharged polar side chain: Ser, Thr, Asn,
Glu, Tyr and Cys.
3. Charged polar side chains are positively or negatively charged. Five amino
acids have charged side chains: Lys, Arg, His, Asp, Glu. The side chains of the
basic amino acids are positively charged al physiological pH values.
The chemical and physical properties of amino acids side chains also govern the chemical
reactivity of the polypeptide.
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Acid-base properties
When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion: can act as either an
acid (proton donor) or a base (proton acceptor).
Amino Acids Have Characteristic Titration
Curves. The amino acids have two or, for those
with ionizable side chains, three acid-base
groups. At low pH values, both acid-base
groups of Gly are fully protonated, and in the
course of titration with NaOH, Gly loses two
protons in the stepwise fashion characteristic
of a polyprotic acid.
The pH at which a molecule carries no net
electric charge is known as its isoelectric point,
pI.
pI = ½ (pK1 + pK2)
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Stereochemistry
With the exception of Gly, all the amino acids recovered from polypeptides are optically actives: they rotate the
plane of polarized light. The C atoms of the amino acids are asymmetric centers or chiral centers. Chiral centers
give rise to enantiomers which are chemically or physically indistinguishable by most techniques.
All amino acids derived from proteins have the L stereochemical configuration; that is, they all have the same
relative configuration around their C atoms. The importance of stereochemistry in living systems is also a concern
of the pharmaceutical industry. Many drugs are chemically synthesized as racemic mixtures, although only one
enantiomer has biological activity.
Cys, Trp and Met are rare amino acids in
proteins
Leu, Ser, Lys and Glu are the most
abundant amino acids, totaling 32% of all
the amino acids residues in a typical
protein
Chemical modification of the amino
acid side chains during of after
synthesis of a polypeptide chain give
more than 100 different amino acids.
Biologically active amino acids. Amino
acids and their derivatives often function
as chemical messengers for
communication between cells: GABA and
dopamine are neurotransmitters.
Histamine is a potent local mediator of
allergic reactions. Glutathione, an
ubiquitous tripeptide (Glu-Cys-Gly), plays
a role in cellular metabolism.
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Polypeptide diversity
Like all polymeric molecules, proteins can be described in terms of levels of
organization, in this case, their primary, secondary, tertiary, and quaternary
structures.
A protein’s primary structure is the amino acid sequence of its polypeptide chain,
or chains if the protein consists of more than one polypeptide.
The primary structure of bovine insulin
The theoretical possibilities for polypeptides are unlimited. For a protein of n
residues, there are 20n possible sequences.
Actual polypeptides are somewhat limited in size and composition. In
general, proteins contain at least 40 residues or so; polypeptides smaller than that
are simply called peptides.
The vast majority of polypeptides
contain between 100 and 1000
residues.
Multisubunit proteins contain several identical and/or nonidentical chains called subunits.
Some proteins are synthesized as single polypeptides and later are cleaved into two or
more chains that remain associated (insulin).
The size range probably reflects the optimization of several biochemical processes.
Polypeptides are subject to severe limitations on amino acid composition (Cys, Trp and Met
and Hys are rare amino acids in proteins; Leu, Ser, Lys and Glu are the most abundant amino
acids)
Each amino acid has characteristic chemical and physical properties, its presence at a
particular position in a protein influences the properties of that protein.
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Protein evolution
Protein sequence evolution. The primary structures of a given protein from related
species closely resemble one another.
Cytochrome c is an evolutionarily conservative protein; that is, its sequence has undergone
only modest evolutionary changes.
Sequence comparisons provide information on protein structure and
function. In general, comparisons of the primary structures of homologous proteins indicate
which of the protein’s residues are essential to its function, which are less significant, and
which have little specific function.
Constructing phylogenetic trees. The
sequences of homologous proteins can be
analyzed by computer to construct a phylogenetic
tree, a diagram that indicates the ancestral
relationship among organisms that produce the
protein.
Phylogenetic tree of cytochrome c
Proteins evolve at characteristic rates. The rate
at which mutations are accepted into a protein depends
on the extent to which amino acid changes affect the
protein’s function.
Rates of evolution of four proteins
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Levels of protein structure
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Protein secondary structure
Describes the local spatial arrangement of the polypeptides’ backbone atoms
disregarding the conformation of its side chains.
The peptide group has a rigid planar structure due to the resonance that gives the
peptide bond a ~ 40% double-bond character
Peptides, with few exceptions, assume a
trans conformation with adjacent Cα on
opposite sides of the peptide bonds
joining them
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Torsion angles between peptide groups describe polypeptide chain
conformations. The backbone or main chain of a protein refers to the atoms that
participate in peptide bonds, ignoring the side chains of the amino acid residues.
The conformation of the backbone can be described by the torsion angles around
the CN bond () and the CC bond () of each residue.
Φ and Ψ are called torsion or dihedral angles
Φ and Ψ are 180o when the
polypeptide is in its fully
extended conformation. Their
values increase clockwise,
when viewed from the Cα atom
Torsion angles of the polypeptide backbone
The configuration of the polypeptide
backbone is further limited by the steric
interference between the carbonyl (C=O)
of one peptide group and the amide (N-H)
of the next
The number of total possible configurations of the polypeptide backbone are
greatly reduced
Extended conformation of a polypeptide
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The Ramachandran diagram indicates allowed conformations of polypeptides
For Pro Φ is limited to ~ -60o
because R = 5 member ring to N
For Gly, the permissible range
of Φ and Ψ is a lot larger due to
its small overall hinderance (R =
H), and no Cβ
The sterically allowed values of Φ and Ψ can be calculated. Most areas in the
diagram represent forbidden conformations of a polypeptide chain. Only three
regions small regions of the diagram are physically accessible to most residues.
for Φ and Ψ
The Ramachandran diagram
left-handed helix
right-handed helix
sheet
Regular secondary structure: the helix and the sheet. A few elements
of protein secondary structure are so widespread that they are immediately
recognizable in proteins with widely differing amino acid sequences. Both the helix
and the sheet are called regular secondary structures because they are
composed of sequences of residues with repeating Φ and Ψ values.
The α-helix: discovered in 1951 by L. Pauling
Φ = -60o Ψ = -50o
1. The backbone is coiled into a rod-like helix
2. The helix has a right-handed screw sense
3. There are 3.6 residues per turn of helix and a pitch of 5.4 Å
4. The rise per residue is 1.5 Å
5. The carbonyl of residue n is hydrogen bonded to amide of
residue n+4. This results in a strong hydrogen bond that has
N....O distance of 2.8 Å
6. R groups project downward and outward away from the helix
axis. The core of the helix is tightly packed.
7. Avg. length of α-helices in proteins is ~12 residues The helix
5.4 Å
2.8 Å
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Sheets: in 1951, Pauling and Corey postulated the existence of the sheet.
1. Main chain is almost fully extended, and
not coiled
2. Hydrogen bonding occurs between
adjacent polypeptide chains
3. Adjacent strands can be parallel or anti-
parallel
4. They have a rippled or pleated edge-on
appearance (like an accordion). The R
groups on each polypeptide chain
alternately extend to opposite side of the
sheet and are in register on adjacent
chains.
5. The length for residue is now ~ 3.5 Å and
there are two residues per turn
Φ ≈ -120o → -140o
Ψ ≈ +110o → +140o
(↑↑) (↑↓)
4.8 Å
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6. The distance between strands is ~4.8 Å
7. In proteins, β-sheets contain an avg of ~6 strands with
~6 residues on avg
8. β-sheets exhibit a pronounced right-handed twist when
viewed along the strands (i.e. bovine carboxypeptidase A)
Diagram of a sheet in bovine carboxypeptidase A
Pleated appearance of a sheet
Connections between adjacent strands in sheets
Connections between adjacent strands in sheets
Nonrepetitive protein structure. A significant
portion of a protein’s structure may also be irregular
or unique (coils).
Variations in amino acid sequence as well as the
overall structure of the folded protein can distort the
regular conformations of secondary structural
elements ( bulge, kinks).
Turns and loops: stretches of polypeptide that
abruptly change direction (reverse turns or
bends, loops)
Space-filling model of an loops
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Tertiary structure
Describes the folding of its secondary structural elements and specifies the
positions of each atom in the protein, including those of its side chains.
The structure of proteins can be
determined by X-ray crystallography and
NMR spectroscopy.
The PDB (protein data bank) is a publicly
available data base of all solved protein
structures: http://www.rcsb.org/pdb.
Nearly 30,000 protein structures have
been reported, no two are exactly alike,
but they exhibit remarkable consistencies.
The nonpolar side chains of a globular proteins tend to occupy the protein’s
interior, whereas the polar side chains tend to define its surface.
Despite the huge number of possible sequences, certain arrangements or patterns
of secondary structures appear to repeat over and over.
While some proteins are purely helical (a, E.coli cytochrome b562) or purely
sheets (b, immunoglobulin fold), most are combinations of both (c, lactate
dehydrogenase).
Supersecondary structures and domains
The proteins are represented by their peptide backbones and its corresponding
topological diagram indicating the connectivity of its helices and strands.
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Certain combinations of helix and sheets are called supersecondary
structures or MOTIFS. These occur in many unrelated globular proteins, as
revealed by the thousands of structures solved to date. Motifs may have
functional as well as structural significance.
1. motif: the most common
2. hairpin: anti-parallel strands connected by reverse turns
3. motif: two successive anti-parallel helices, which are packed with their axes
inclined
4. Greek key motif: a hairpin is folded over to form a 4-stranded antiparallel sheet
motif hairpin motif Greek key motif
5. -barrels motif: -sheets rolling up into barrels
-barrels
Helix-loop-helix
6. Helix-loop-helix motifs: helix-turn-helix
(HTH) motif contain two helices that cross at
an angle of ~ 120º
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Common structural motifs in DNA-binding proteins include the helix-turn-helix motif
in prokaryotic repressors, and zinc fingers, Leucine zippers, and basic helix-loop-
helix motifs in eukaryotic transcription factors.
Leucine zipper motif: two -helices “zipper” together by Leucine residues every
7th residue in a repeating amino acid sequence
The GCN4 leucine zipper motif and its X-ray structure in complex with its target DNA
These motifs are incorporated into even larger structures.
Motifs may have functional as well as structural significance. For example, /
barrel, four helix bundle, / saddle (combinations of , Rossmann fold often
acts as a nucleotide-binding site), / sandwich
Domains are structurally independent units that have the characteristics of a
small globular protein.
1. Compact globular structures
2. Typical size is 100-200 amino acid residues,
and the average diameter ~ 25 Å
3. Two or more often connected by a loop or a
hinge.
4. Consist of two or more layers of secondary
structural elements
5. Often the domains seem to possess
specialized functions
6. In multidomain proteins, ligands or substrates
bind in the cleft (groove) between two domains The two domain protein flyceraldehyde-3-phosphate dehydrogenase
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Three-dimensional structures of c-type cytochromes
Protein Families
• Collections of proteins that share similar folding pattern and often related functions
• Sometimes little sequence similarity
• Often share a common evolutionary origin
• There are currently several hundred unique protein domains or folds. We expect ~1000
unique (distinct) domains or folds, once all structures are known
• Within a family, structure is closely conserved to a far greater extent than is the case for
sequence
Protein quaternary structure and symmetry
Most proteins (m > 100 kD) consist of more than one polypeptide chain. These
polypeptide subunits associate with a specific geometry. The spatial arrangement of
these subunits is known as a protein’s quaternary structure.
1. Subunits usually associate noncovalently. The contact regions between subunits
resemble the interior of a single-subunit protein.
Hb: dimer of protomers
Degrees of Complexity:
1. Dimer of identical subunits: Alcohol Dehydrogenase
2. Molecules consist of 1 or 2 copies of several subunits: Hemoglobin
3. Multi-subunit with fixed total size and stoichiometry: E. coli Pyruvate
Dehydrogenase with 60 subunits
4. Multi-subunit with fixed stoichiometry, but varying size: microtubules, and
tubulin
2. Subunits are symmetrically arranged. In most oligomeric protein, the protomers
are arranged symmetrically. Each polypeptide chain has these properties:
Asymmetric
Cs are asymmetric
No inversion or mirror symmetry
Quaternary structures can only have rotational symmetry
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Some symmetries of oligomeric proteins
Protein stability and dynamics
Native proteins structures are only slightly more stable than their denatured forms.
The hydrophobic effect is the primary determinant of protein stability.
Hydrogen bonding and ion pairing contribute relatively little to a protein’s stability.
Proteins are flexible and rapidly fluctuating molecules whose structural mobilities are
functionally significant. Theoretical calculations indicate that a protein’s native structure
probably consists of a large collection of rapidly interconverting conformations that have
essentially equal stability.
Conformation flexibility (breathing) with structural
displacement of up to ~ 2 Å, allows small molecules
to diffuse in and out of the interior of certain proteins.
Molecular dynamics of myoglobin. Several
“snapshots“ of the protein calculated at intervals of
5 x 10-12
s.
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Protein folding
The sequence of protein determines its three-
dimensional structure, which determines its function. In
short, function derives from structure; structure derives
from sequence.
Because protein function derives from protein structure,
newly synthesized proteins must fold into the correct
shape to function properly.
The planar structure of the peptide bond limits the
number of conformations a polypeptide can have.
The amino acid sequence of a protein dictates its
folding into a specific three-dimensional conformation, the
native state. Proteins will unfold, or denature, if treated
under conditions (heat, extreme pHs, detergents,
chaotropic agents or denaturants) that disrupt the
noncovalent interactions stabilizing their three-
dimensional structures.
Proteins fold to their native conformations via directed
pathways in which small elements of structure coalesce
into large structures.
Hypothetical protein folding pathway
Energy-entropy diagram for protein folding
Protein folding in vivo occurs with assistance from chaperones, which bind to nascent
polypeptides emerging from ribosomes and prevent their misfolding.
Some neurodegenerative diseases (Alzheimer’s disease, the transmissible
spongiform encephalopathies, the amyloidoses) are caused by aggregates of proteins
that are stably folded in an alternative conformation.
Alzheimer’s disease is characterized by the
formation of insoluble plaques composed of
amyloid protein
A cluster of partially proteolyzed prion rods labeled
with colloidal gold beads coupled to anti-PrP
antibodies.
A model of an amyloid fiber. The
arrowheads indicate the path of the
strands.
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Protein function
The function of nearly all proteins depend on their ability
to bind other molecules (ligands).
The specificity of a protein for a particular ligand refers to
the preferential binding of one or a few closely related
ligands.
The affinity of a protein for a particular ligand refers to the
strength of binding, usually expressed as the dissociation
constant Kd.
Proteins may be regulated at the level of protein
synthesis, protein degradation, or the intrinsic activity of
proteins through noncovalent or covalent interactions.
In allostery, the noncovalent binding of one ligand
molecule (substrate, activator or inhibitor), induces a
conformational change that alters a protein’s activity or
affinity for other ligands.
In multimeric proteins, such as Hb, that bind multiple
identical ligand molecules, the binding of one ligand may
increase or decrease the binding affinity for subsequent
ligand molecule (cooperativity)
Protein-ligand binding of antibodies
Protein structure and function
ENZYMES
Mb and Hb
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ENZYMES are catalytic proteins that
accelerate the rate of cellular reactions by
lowering the activation energy and
stabilizing transition-state intermediates.
Enzymes are highly efficient and specific catalysts
An enzyme active site comprises
two functional parts: a substrate-
binding site and a catalytic site.
Active site of the enzyme trypsin Active site of the enzyme trypsin
V0 = Vmax
[S]
[S] + KM
An enzyme’s active site binds substrates and carries out catalysis
The Michaelis constant KM is a
rough measure of the enzyme’s
affinity for converting substrate into
product.
The maximal velocity Vmax is a
measure of its catalytic power.
1913, Leonor Michaelis y Maud Menten
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Kinetic data can be plotted in double-reciprocal form to determine KM and Vmax
Enzyme Inhibition
Many substances alter the activity of an enzyme by combining with it in a way that
influences the binding of substrate and /or its turnover number. Substances that
reduce an enzyme’s activity in this way are known as inhibitors.
Reversible inhibitors reduce an enzyme’s activity by binding to the substrate-
binding site (competitive inhibition), to the enzyme-substrate complex
(uncompetitive inhibition), or to both the enzyme and the enzyme-substrate
complex (mixed inhibition).
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The degree of competitive inhibition
depends on the fraction of enzyme
that has bound inhibitor.
Competitive inhibition is the principle
behind the use of ethanol to treat
methanol poisoning.
ADH
Comparing the KI values of competitive inhibitors with different structures can provide
information about the binding properties of an enzyme’s active site and hence its
catalytic mechanism. For example, to ascertain the importance of the various
segments of an ATP molecule for binding of the active site of an ATP-requiring
enzyme.
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The HIV protease inhibitors have been designed to mimic the enzyme’s transition
state and thus bind to the enzyme with high affinity.
Noncovalent binding permits allosteric, or cooperative,
regulation of proteins
One of the most important mechanisms for regulating protein function is through
allosteric interactions.
+ -
-Cooperatividad + -
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Mb
The heme group
Regulatory Strategies: Enzymes and the oxygen-binding proteins
Myoglobin and Hemoglobin CO, NO and SH2
Myoglobin’s oxigen-binding curve is hyperbolic
In experiments using oxygen as a ligand, it is the partial pressure of
oxygen in the gas phase above the solution, pO2, that is varied,
because this is easier to measure than the concentration of oxygen
dissolved in the solution. The concentration of a volatile substance in
solution is always proportional to the local partial pressure of the gas.
So, if we define the partial pressure of oxygen at [O2]0.5 as P50,
substitution in Equation gives
2.8 torr
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Hemoglobin, a tetramer with pseudo-D2 symmetry, has distinctly
different conformations in its oxy and deoxy states.
Hemoglobin Is a Tetramer with Two Conformations
Oxygen Binds Cooperatively to Hemoglobin
O2 binding to hemoglobin is described
by a sigmoidal (S-shaped) curve. This
permits the blood to deliver much more
O2 to the tissues than if hemoglobin had
a hyperbolic curve with the same p50.
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Myoglobin is a useful model for other binding proteins. Even proteins with multiple
binding sites for the same small molecular, or ligand, generate hyperbolic binding
curves. Moreover, hemoglobin exhibits a sigmoidal oxygen-binding curve, a
diagnostic of a cooperative interaction between binding sites. This permits the blood
to deliver much more O2 to the tissues than if Hb had a hyperbolic curve.
The Hill equation describes hemoglobin’s oxigen-binding curve
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T state (deoxyHb, blue)
R state (oxyHb, red)
Hemoglobin has two conformational states. Perutz formulated a mechanism for Hb
oxygenation: T and R.
Hemoglobin Also Transports H+ and CO2
CO2 promotes oxigen dissociation from Hb through the Bohr effect, BPG
decreases hemoglobin’s oxigen affinity by binding to deoxyhemoglobin
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The O2 affinity of Hb increases with increasing pH.
lungs
tissue
Bohr effect
This phenomenon is known as the Bohr effect after Christian Bohr (father of the physicist Niels Bohr),
who first reported it in 1904.
Oxygen Binding to Hemoglobin Is
Regulated by 2,3-Bisphosphoglycerate
BPG binds tightly to deoxyhemoglobin but only
weakly to oxyhemoglobin. The presence of BPG in
mammalian erythrocytes therefore decreases
hemoglobin’s oxygen affinity by keeping it in the
deoxy conformation.
High-altitude adaptation is a complex process that involves
Increases in the number of erythrocytes and the amount of
hemoglobin per erythrocyte.
High-Altitude Adaptation
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Two models that account for cooperative ligand binding have
received the most attention. One of them, the symmetry model
of allosterism, formulated in 1965 by Jacques Monod, Jeffries
Wyman, and Jean-Pierre Changeux, is defined by the following
rules:
Hemoglobin Is a Model Allosteric Protein.
1. An allosteric protein is an oligomer of symmetrically
related subunits (although the and subunits of
hemoglobin are only pseudosymmetrically related).
2. Each oligomer can exist in two conformational states,
designated R and T; these states are in equilibrium.
3. The ligand can bind to a subunit in either conformation.
Only the conformational change alters the affinity for
the ligand.
4. The molecular symmetry of the protein is conserved
during the conformational change. The subunits must
therefore change conformation in a concerted manner;
in other words, there are no oligomers that
simultaneously contain R- and T-state subunits.
The molecular defect in sickle-cell hemoglobin was not identified until 1956, when Vernon Ingram
showed that hemoglobin S contains Val rather than Glu at the sixth position of each chain. This was
the first time an inherited disease was shown to arise from a specific amino acid change in a protein.
A Single Amino Acid Change Causes Sickle-Cell Anemia.
The regions of equatorial Africa where malaria is a
major cause of death coincide closely with those
areas where the sickle-cell gene is prevalent,
thereby suggesting that the sickle-cell gene
confers resistance to malaria.
How does it do so? Plasmodia increase the acidity
of infected erythrocytes by 0.4 pH units. The lower
pH favors the formation of deoxyhemoglobin via the
Bohr effect, thereby increasing the likelihood of
sickling in erythrocytes that contain hemoglobin S.
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An alternative to the symmetry model is the sequential model of allosterism, proposed by Daniel
Koshland. According to this model, ligand binding induces a conformational change in the subunit to which
it binds, and cooperative interactions arise through the influence of those conformational changes on
neighboring subunits. The conformational changes occur sequentially as more ligand-binding sites are
occupied
Molecular Chaperones Assist Protein Folding
Molecular chaperones are essential proteins that bind to unfolded and partially folded
polypeptide chains to prevent the improper association of exposed hydrophobic segments
that might lead to non-native folding as well as polypeptide aggregation and precipitation.
X-Ray structure of the GroEL–GroES–(ADP)7 complex.
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METHODS
PROTEIN PURIFICATION, DETECTION AND ANALYSIS
Protein purification and analysis
Purification is an all but mandatory step in studying macromolecules, but it is not
necessarily easy.
The first step in the isolation of a protein or other biological molecule is to get it out of the
cell and into solution. Many cells require some sort of mechanical disruption to release their
content. If the target protein is associated with a lipid membrane, a detergent may be used to
solubilize the lipids and recover the protein.
Factors to be controlled at all stages of a purification process: pH, temperature, presence
of degradative enzymes, adsorption to surface, long-term storage.
Proteins are purified by fractionation procedures which depend on the protein
characteristics are based on:
protein characteristic purification procedure
solubility salting out
ionic charge ion exchange chromatography, electrophoresis, isoelectric focusing
polarity hydrophobic interaction chromatography
size gel filtration chromatography, SDS-PAGE, ultracentrifugation
binding specificity affinity chromatography
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Assaying proteins
purifying a substance requires some means for quantitatively detecting it.
An assay must be specific for the target protein, highly sensitive, and
convenient to use.
Among the most straightforward protein assays are those for enzymes that
catalyze reactions with readily detected products. Proteins that are not
enzymes can be detected by their ability to specifically bind certain
substances or to produce observable biological effects.
Immunochemical procedures are among the most sensitive of assay
techniques.
Radioimmunoassay (RIA), the protein is indirectly detected by determining
the degree to which it competes with a radioactively labeled standard for
binding the antibody.
Enzyme-linked immunosorbent assay (ELISA) has many variations, one of
which is diagrammed below.
The concentration of a protein in solution can be measured by absorbance
spectroscopy (Beer-Lambert’s Law; 50 to 100 mg per mL).
The Bradford assay provides a direct measure of the amount of protein
present (1 mg of protein per mL).
ELISA
UV absorbance spectra
Separation techniques
ELECTROPHORESIS CENTRIFUGATION
Centrifugation is used for two basic purposes: as a
preparative technique to separate one type of
material form others and as an analytical
technique to measure physical properties of
macromolecules (MW, density, shape and Keq).
SDS-PAGE separates proteins purely by gel filtration
effects, that is, according to molecular mass. The
relative mobilities of proteins vary ~ log (molecular
mass). The separated bands may be visualized in the
gel by an appropriate technique (Abs, radioisotopes).
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Separation techniques
Liquid chromatography separates proteins on the
basis of their rates of movement through a column
packed with spherical beads. Binding and elution
of the proteins often depend on the salt
concentration and pH.
CHROMATOGRAPHY PROTEIN SEQUENCING
The protein must be broken down into fragments
small enough to be individually sequenced, and
the primary structure if the intact protein is then
reconstructed from the sequences of overlapping
fragments (e.g. mass spectroscopy).
Protein conformation is determined by sophisticated physical methods
X-RAY CRYSTALLOGRAPHY NMR SPECTROSCOPY
X-ray crystallography provides the most detailed structures but requires protein crystallization. Only relatively
small proteins are amenable to NMR analysis. Cryoelectron microscopy is most useful for large protein
complexes, which are difficult to crystallize.
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PROTEOMICS
Proteomics is the systematic study of the amounts
(and changes in the amounts), modifications,
interactions, localization, and functions of all or subsets
of all proteins in biological systems at the whole-
organism, tissue, cellular, and subcellular levels.
Proteomics provides insights into the fundamental
organization of proteins within cells and how this
organization is influenced by the state of the cell (e.g.
differentiation into distinct cell types; response to stress,
disease, and drugs)
A wide variety of techniques are used for proteomic
analysis, including two-dimensional gel electrophoresis,
density gradient centrifugation, and mass spectroscopy.
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Allosteric mechanisms can cause large changes in enzymatic activity.
Enzyme: aspartate transcarbamoylase (ATCase) from E. coli.
Function: it catalizes the formation of N-carbamoyl aspartate, the first step
unique to the biosynthesis of pyrimidines.
Both substrates bind cooperatively to the enzyme
ATCase is allosterically inhibited by CTP (pyrimidine nucleotide), and its
allosterically activated by ATP (purine nucleotide). CTP is an example of a
feedback inhibitor, since it inhibits an earlier step in its own biosynthesis. ATCase
consists of separable catalytic (c3) subunits (which bind the substrates) and
regulatory (r2) subunits (which bind CTP and ATP).
Aspartate Transcarbamoylase Is Allosterically inhibited by the End
Product of Its Pathway
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E. coli ATCase (300 kDa) has the subunit composition c6r6 where c and r represent its
catalytic and regulatory subunits. On binding substrates, the c3 subunits of the c6r6 enzyme
move apart and reorient themselves. This allosteric transition is highly concerted, as
postulated by the Monod-Wyman-Changeux (MWC) model. All subunits of an ATCase
molecule simultaneously interconvert from the T (low-affinity) to the R (high-affinity) state.
The activity of ATCase is increased by ATP an decreased by CTP, which alter the conformation of
the catalytic sites by stabilizing the R and the T states of the enzyme, respectively.
Regulatory dimers (yellow)
Catalytic trimer (red)
Catalytic trimer (blue)
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PALA is a potent competitive inhibitor of ATCase; it binds to and blocks the active sites. The
structure of the ATCase–PALA complex reveals that PALA binds at sites lying at the
boundaries between pairs of c chains within a catalytic trimer.
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