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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.