The three-dimensional structure of proteins

levels of structures in proteins

Primary – simply the aa sequence
Secondary – describes how segments of the peptide backbone
orient into a regular pattern
alpha helix; beta sheet
Tertiary – describes how the entire protein molecule coils into
an overall 3D shape
ex: myoglobin
Quaternary – describes how different protein molecules come
together to yield large aggregate structures
ex: hemoglobin

Proteins – many different conformations (3D structures) are possible

 Native conformation – one or a few of these
which have biological activity

Four levels of structures of proteins:

1. Primary structure – the order in which the aa’s are covalently linked together
- the first step in specifying the 3D structure of a protein
2. Secondary structure – the arrangement in space of the atoms in the
peptide backbone
- alpha helix and beta pleated sheet
3. Tertiary structure - includes the 3D arrangement of all the atoms in the
protein, including those in the side chains and in any
prosthetic grp (grps of atoms other than aa’s)
4. Quaternary structure – applicable to proteins w/c consist of multiple
polypeptide chains called sub-units
- arrangement of sub-units wrt one another
- interxn b/w subunits mediated by non-covalent
interxns such as H-bonds, electrostatic interxns,
and hydrophobic interxns

The primary structure of proteins - the aa sequence

- determines the 3D structure w/c in turn det the properties
- correct 3D structure needed for correct functioning of protein
Example:
sickle-cell anemia – a genetic disease wherein RBC cannot bind O2 efficiently
- RBC assumes a sickle shape, hence the name of the disease
- sickled cells tend to be trapped in small blood vessels,
cutting off circulation, and thereby causing organ damage
- RBC contains the protein hemoglobin
- there was a change in one aa residue in the sequence of
the primary structure of hemoglobin.
Site-directed mutagenesis – a molecular biology technique
- possible to replace any chosen aa in a protein
with another specific aa
The results of aa substitution can range from negligible effects to complete loss of activity

The secondary structure of proteins

- hydrogen-bonded arrangement of the backbone of the polypeptide chain
- two bonds with reasonably free rotation:
- b/w α-C and amino-N
- b/w α-C and carboxyl-C
- the peptide chain backbone can be visualized as a series of playing cards, each card representing
a planar peptide group.
- the cards are linked at opposite corners by swivels, representing the bonds having freedom of
rotation

angle Φ (phi) designate C-N bond rotation

angle ψ (psi) designate C-C bond rotation
--- Ramachandran angles (G.N. Ramachandran)
The α-helix
- stabilized by H bonds parallel to the helix axis w/in the backbone of a single polypeptide chain
- counting from the N-terminal end, the C-O grp of each aa is H-bonded to the N-H grp of the aa
that is 4 residues away from it in the covalently bonded sequence.
- allows linear arrangement of the atoms involved in the H bonds, thus giving the bonds
maximum strength and makes the helical conformation very stable
- pitch of helix = 5.4 Å
= 0.54 nm = 540 pm
- The linear distance b/w corresponding points on successive turns
Proteins have varying amts of α-helical structures varying from:
a few percent to
100 %
Factors that can disrupt the α-helix:
1. proline – creates a bend in the backbone because of its cyclic structure
- cannot fit into the α-helix because:
a. rotation around the bond b/w N and α-C is severely restricted
b. proline’s α-amino grp cannot participate in intra-chain
H-bonding
2. electrostatic repulsion
proximity of sev charged grps of the same sign
grps of +ly charged lys and arg
grps of –ly charged glu and asp

The β-sheet
- peptide backbone is almost completely extended
- intrachain H-bonds on parts of a single chain that doubled back on itself
- interchain H-bonds
Parallel pleated sheet – formed if the peptide chains run in the same direction
-- Aligned N- and C terminal ends

Antiparallel pleated sheet – in opposite directions

H-bonding between peptide chains gives rise to a zigzag structure hence, the name pleated sheet.
H-bonds are perpendicular to the direction of the protein chain, not parallel to it as in the α-helix.
 3D form of the antiparallel β-pleated sheet arrangement.
The chains do not fold back on each other but are in a fully extended conformation.

Irregularities in regular structures

Some structures in proteins break up the regular nature of the α-helix:

β-bulge – a region of irregularity in a β-sheet, where the normal pattern of H bonding is disrupted by the
insertion of an extra residue

Classic bulge Wide bulge G-1 bulge – occur only in

antiparallel sheet
Protein folding requires that the peptide backbones and the 2o structures be able to change directions.
Reverse turns – marks a transition between one 2o structure and another
when a polypeptide chain changes direction
The ff aa’s are frequently encountered in reverse turns:
1. glycine – single H of side chain prevents crowding
2. proline – has the correct geometry for a reverse turn

In type 1, any aa can be involved.

Supersecondary structures and domains

- produced when α-helix, β-sheet, and other 2o structures are combined in many ways as the pp
chain folds back on itself
- these include:

1. βαβ unit – two parallel strands of β-sheet are connected by a stretch of α-helix

2. αα unit – helix-turn-helix
- two antiparallel α-helices
- energetically favored contacts exist b/w the side chains in the α-helices
3. β- meander – an antiparallel sheet is formed by a series of tight reverse turns connecting stretches of
the pp chain

4. Greek key – a kind of antiparallel sheet wherein the pp chain doubles back on itself in a pattern similar
to a decorative design found on pottery from the classical period

5. Motif – a repetitive supersecondary structure

- sometimes called module

Growth factor
complement-control
Immunoglobulin globle
protein molecule
molecule

fibroconnectin type I
module

Kringle module
6. β-barrel arrangements

a. linked series of β-meanders – in the protein rubredoxin from Clostridium pasteurianum

b. Greek key pattern – in human prealbumin

c. β-barrel involving alternating βαβ units – in triose phosphate isomerase from chicken muscle

top and side views of the pp backbone arrangement in triose phosphate isomerase showing that the α-
helical sections lie outside the actual β-barrel
Collagen – a component of bone and connective tissue
- most abundant protein in vertebrates
- organized in water-insoluble fibers of great strength

Myoglobin – first protein for which the complete 3o structure was determined
- consists of 153 aa residues and a heme grp (prosthetic grp)

- Has 8 α-helical regions

- there are no β-pleated sheet regions
- H-bonding in the pp backbone stabilizes the α-helical regions
- aa side chains are also involved in hydrogen bonds
- polar residues are on the exterior of the molecule
 - two polar his in interior are involved in interxns with heme grp and bound O2 thus, play an impt
role in protein fn (oxygen-storage protein)
- interior of the protein mostly cont nonpolar aa residues
- the planar heme grp fits into a hydrophobic pocket and held in position by hydrophobic
attractions b/w the heme’s porphyrin ring and the nonpolar side chains
- the apoprotein (the pp chain alone) is not as tightly folded as the complete molecule

The structure of the heme grp.

Four pyrrole rings are linked by bridging grps to form a planar porphyrin ring.
Several isomeric porphyrin rings are possible depending on arrangement of side chains.
The porphyrin isomer found in heme is protoporphyrin IX.
Addition of iron to protoporhyrin IX produces the heme grp.

The oxygen-binding site of myoglobin.

- the porphyrin ring occupies 4 of the 6 coordination sites of the Fe (II)
- His F8 occupies the 5th coordination site of the iron
- oxygen is bound at the 6th coordination site of the iron
- His F7 lies close to the oxygen
Combination of both heme and protein is needed to bind O2.
- in the absence of the protein, the iron of the heme grp can be oxidized to Fe(III)
- oxidized heme will not bind oxygen

Two types of proteins according to 3D shape:

Schematic
Schematic diagram
diagram

a. Fibrous proteins - insoluble Computer

(collagen, keratin) generated
model

b. Globular proteins – soluble

(myoglobin, hemoglobin
Two types of proteins according to composition:
Simple proteins – those that yield only aa’s on hydrolysis
- blood serum albumin
Conjugated proteins – yield other compounds such as
carbohydrates, fats, or nucleic acids in
addition to aa’s on hydrolysis

Tertiary structure
--- The forces that determine the tertiary structure of proteins are the same forces that act on all
molecules, regardless of size, to provide maximum stability. These include:

a. hydrophilic (water-loving) interactions of the polar side chains on acidic and basic aa’s
- acidic or basic aa’s w/ charged side chains tend
to congregate on the exterior of the protein
where they can be solvated by water
b. hydrophobic interxns of neutral, nonpolar side chains
- they tend to congregate on the hydrocarbon-like
interior of a protein molecule, away from the
aqueous medium
c. formation of disulfide bridges b/w cysteine residues
d. formation of H-bonds b/w nearby aa residues
e. development of ionic interxns, called “salt bridges” b/w +ly and –ly
charged sites on various aa side chains within the protein

Forces that stabilize the tertiary structure of proteins.

The noncovalent interxns that maintain the 3o structure of proteins are weak
--- can be disrupted easily and leads to unfolding of protein (denaturation)

Disruption of 3o structure can be due to:

Heat – temp inc favors vibrations w/in the molecule and the E of these vibrations can disrupt 3 o
structure
High or low extremes of pH – can lead to loss of some of the charges on protein and thus, electrostatic
interxns that stabilize native, active form are drastically reduced

Detergents – tend to disrupt hydrophobic interxns

- charged detergents disrupt electrostatic interxns

Urea
Guanidinium hydrochloride
-- form H-bonds with the protein, disrupt hydrophobic interxns w/in protein

Β-mercaptoethanol – reduces disulfide bridges to two sulfhydryl grps

- urea added to facilitate protein unfolding to inc
accessibility of the disulfides to the RA

Denaturation and refolding in RNAse.

The protein can be completely denatured by the actions of heat and urea.
When denaturing conditions are removed, activity is recovered.
Quiz
Name the different factors that would disrupt the tertiary structure of a protein.

Quaternary structure – a property of proteins that consist of more than one pp chain (2 or more than
12) w/c may be identical or different
- ea chain is called a subunit
- oligomer (dimer, trimer, or tetramer)
- chains interact w/ ea other noncovalently via
electrostatic interactions
H bonds
hydrophic interxns

The structure of hemoglobin (α2β2).

- A tetramer consisting of four pp chains.
- two α-chains and two β-chains

In hemoglobin molecule,
- four molecules of oxygen can bind
- binding of oxygen to hemoglobin exhibits positive cooperativity
(but not myoglobin)

Means that when one molecule of oxygen is bound, it becomes easier for the next molecule to bind.

Curve is hyperbolic
-A steady rise is observed until complete satn is approached and the curve levels off
Functions in oxygen storage in the muscle.
- Must bind at low P
Curve is sigmoidal
-Indicates that binding of the first O2 facilitates that of the 2nd w/c facilitates that of the 3rd w/c in turn
facilitates that of the 4th
-“cooperative binding”
-Functions in oxygen transport.
Must bind/release depending on conditions.
- In the alveoli of lungs, P=100 torr, protein must bind for transport to tissue. At this P, protein is
satd with O2.
- In capillaries of active muscles, P=20 torr, <50% satn of protein, oxygen is easily given up
because it is greatly needed.

In an actively metabolizing tissue,

oxyhemoglobin Deoxyhemoglobin
 oxyhemoglobin deoxyhemoglobin
There is much less room at the center of oxyhemoglobin.

Some biochemical applications:

Inborn errors of metabolism – mutations leading to deficiencies in enzymes
- involve defects in the DNA of the affected individuals
phenylketonuria (PKU) – deficiency in the enzyme that catalyzes the conversion of phenylalanine
to tyrosine
- thus, leads to the accumulation of phenylpyruvate, a phenyl ketone.

phenylalanine
transaminase
Phenylalanine
hydroxylase Phenylpyruvate (a phenyl ketone)
enzyme deficiency in
PKU
phenyllactate

tyrosine
phenylacetate

Phenylpyruvate – a phenyl ketone

- causes mental retardation by interfering with the conversion of pyruvate to acetyl-CoA
in the brain
- accumulation in brain cells results in osmotic imbalance in which water flows into the
brain cells. Cells thus expand in size and hinders normal development of the brain.
- easily detected in U.S. in newborns and treated by a
modified diet  limited phe and tyr is supplemented
Aspartame – artificial sweetener
- carries the warning of phe content
Alatame – aspartame substitute
- does not contain phe
- thus, has benefits of aspartame without danger assocd with phe

oxytocin vasopressin
-- Peptide hormones
-- Released by the action of the hypothalamus on the posterior pituitary and transported by the blood to
specific receptors

Oxytocin – induces labor during pregnancy and controls contraction of uterine muscle
- stimulates milk flow in nursing mother
Vasopressin – controls blood pressure by regulating the contraction of smooth muscle

Glycine – assists in the disposal of certain molecules by making them more water soluble and thus
excreted in the urine
benzoic acid + glycine  hippuric acid (as hippurate)
glycine + cholic acid  glycocholate (one of major bile salts
 important in fat digestion)
Methionine – a derivative of this is S-adenosylmethionine (SAM; source of methyl grp in methylation
rxns)
- another derivative is ethionine, a potent poison (cont an ethyl grp and thus an ethyl grp is
transferred rather than methyl)
Glutamic acid – a derivative is monosodium glutamate (MSG; a flavor enhancer)
- MSG causes physiological rxns in some people (chills, dizziness, hedaches)  referred to
as Chinese restaurant syndrome bec many Asian food contain sig amts of MSG

β-alanine – found in the vitamin pantothenic acid and is an important part of the enzyme cofactor
Coenzyme A

Histidine – a derivative is histamine wherein the acid grp in his is removed

histamine
-- A potent vasodilator – inc the diameter of blood vessels and results in swelling and stuffiness that are
associated with a cold.
 most cold medications contain antihistamines to overcome this stuffiness

Arginine – involved in the urea cycle, a series of rxns important in the use of nitrogen by living organisms
arginine  ornithine  urea

asparagine
glutamine
• Involved in the detoxification of ammonia in animals
• Play a role in nitrogen storage in plants

Aspartame – methyl ester derivative of the dipeptide asp-phe

- about 200x sweeter than sugar
- marketed under the trade name NutraSweet

Prion – small (28-kD) protein

- causative agent of mad-cow disease and related diseases
scrapie in sheep
spongiform encephalopathy in humans
Prions are glycoproteins found in the cell membranes of nerve tissue.
– when the normal form folds into an incorrect form  diseases

a. Normal structure (large percentage of α-helix)

b. Abnormal structure (have more β-pleated sheets)