Protein Function

Structure determines function

Figure 4.11 Models of myoglobin structure.

Myoglobin
First protein structure solved by X-ray
cystallography
Single peptide chain
Contains eight helices numbered A
through H
Contains a heme moeity in a pocket or
crevice
An oxygen-binding protein

Heme

Heme is a prosthetic group.

Prosthetic group = organic
molecule bound to protein
that aids protein function
Heme is a porphyrin that
chelates iron for oxygen
transport.

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Oxygen Binding to MbG

The heme molecule sits in a hydrophobic pocket
formed mainly by the E and F helices
When O2 binds to the heme of MbG, the Fe2+
moves back toward the plane of the heme molecule
bringing the proximal histidine, His F8 with it
This movement places the O2 close to the distal
histidine, His E7, which coordinates with the
bound O2

Figure 4.20 Oxygen binding to the heme group of myglobin.

Oxygen Binding in Myoglobin

Depends on oxygen concentration
Mb + O2 MbO2
From the equation, we can write a
dissociation constant expression
K = [Mb][O2]/[MbO2]

Oxygen Binding in Myoglobin

The fractional saturation, Y, is defined as
Y = [MbO2]/ [Mb] + [MbO2]
Since [MbO2] = [Mb][O2]/K
Y= ([Mb][O2]/ K)/ [Mb] + [Mb][O2]/K
= ([O2]/ K)/ 1 + [O2]/K
= [O2]/ K + [O2]
Since O2 is a gas, the concentration can be expressed
in partial pressure
Y= pO2 / K + pO2

O2 binds to the heme

group of myoglobin
such that binding is
half-maximal when the
oxygen concentration is
equal to the
dissociation constant.

Hyperbolic data is common in biochemistry

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Hemoglobin
The major oxygen carrying molecule in the
blood of high animals
Human hemoglobin is a tetramer composed
of two pairs of identical subunits
Two chains
Two chains

Each subunit contains a heme molecule

Mb and Hb are only ~18%

identical in primary sequence.

Invariant

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Identical in all

Identical in Hb

Mb and Hb are similar in their

secondary and tertiary
Heme
structures.

Even though
myoglobin and hemoglobin have
only ~18% identical residues,
their secondary and tertiary
structures overlap almost
perfectly when superimposed!

Myoglobin
-Subunit of
Hemoglobin

Hb has quaternary
structure, but Mb
does not.
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Oxygen binds cooperatively to

Dotted line represents
Hb.
O2
binding to myoglobin
(hyperbola).

Solid line represents O2

binding to hemoglobin
(sigmoid).
Note:
Sigmoidal data are
indicative of
cooperativity. Cooperativity:
Binding of O2 to one subunit induces easier binding to
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Cooperativity
Binding of a ligand to one subunit affects
the binding of the ligand to another subunit
Hemoglobin subunits exist in two
conformational states
R which is the oxygen bound or oxy-HbG
T which is the unoxygenated or deoxy-HbG

Binding of the first O2 to the T state is

difficult

Cooperativity
The major changes in the T state and the R
state are found at the 12 and 21
When the first O2 binds to a subunit, the
Fe2+ moves into the plane of the porphyrin
ring
The proximal His F8 moves with the
coordinated Fe2+

Cooperativity
Movement of the tightly packed His F8 causes the
F helix to translate across the heme plane
The movement of the F helix results in a shift of
the 1C-2FG contact one turn along the 1C helix
The inflexibility of the 11 and 22 interface
require that the shift occurs simultaneously at both
12 and 21 interfaces

Figure 4.27 Conformational changes in hemoglobin upon O2 binding.

Figure 4.28 Some of the subunit interactions in hemoglobin.

Bohr Effect and O2 Transport

What is happening biochemically when you breathe?

From
Metabolism
+ H2O
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As pH , O2 affinity

From
Metabolism
+ H2O
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The Bohr Effect

The N-terminal groups of the chains and
two His near the C-terminus of the chains
release H+ when O2 binds
As pH decreases, the oxygen binding
affinity decreases
Important in the tissues
Increases off-loading of oxygen
Provides H+ to help transport CO2

2,3-Bisphosphoglycerate (BPG)
A 3 carbon compound with a carboxyl
group and two phosphate charges
Has an total 5 charge
Binds to positive charges of the central
cavity of deoxy-HbG and stabilizes its
conformation
Is expelled when oxygen binds
Lowers oxygen affinity to allow efficient
off loading of oxygen in the tissues

Fractional Saturation of O2

BPG decreases Hbs O2

affinity.
Lower O2
affinity

BPG binds only to the

tense (deoxy)
conformation of Hb.

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Comparison of HbG and Deoxy-HbG

Allosteric Proteins
HbG is one example of an allosteric protein
The binding of a ligand in one site effects
the binding of a ligand at another site
Ligands binding to the two sites may be the
same or different
Effect may be activating or inhibitory

Structural Protein
Elongated, regular structures
Cytoskeletal- shape and anchoring
Some use ATP and GTP to convert chemical
energy to mechanical energy

Some Important Structural

Proteins
Microfilaments
Actin

Microtubules
Tubulin

Intermediate Fibers
Keratin

Collagen

Actin
Monomeric or G-actin
Globular protein- about 375 residues
A surface cleft binds ATP

Actin polymer or F-actin

Polymer forms so that the ATP-binding cleft of
all the subunits are oriented in the same
direction
A double chain of subunits with each subunit
interacting with four neighboring subunits

Globular actin subunits associate in

a double chain to form a
microfilament.

Polymerization

Actin monomer
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Figure 5.04 Microfilament assembly.

Microfilaments
Microfilaments of actin are dynamic
The (-)end has an exposed ATP site and the
(+)end does not
Polymerization is faster at the (+)end
Most of the ATP sites are occupied by ADP
Polymerization is reversible- at equilbriumtreadmilling
In vivo- ends are capped

Figure 5.05 Microfilament treadmilling.

Microfilaments
Growth in one area is at the expense of
diassembly elsewhere
In vivo, growth occurs where an end has lost
cap
New may occur by branching
Microfilaments can be weakened and
severed
Microfilaments are subject to control by
signaling proteins

Microtubules
Thin, flexible tubes
Shape gives added strength and rigidity to
the structure
Composed of two tubulin monomers

Microtubules
Composed of tubulin
Two monomers, and
Each type is composed of about 450 residues
with 40% homology
Subunits form as dimers
Tubulin core is a 4 stranded sheet and a
6stranded sheet
Each subunit has a nucleotide binding siteGTP

Figure 5.12 Structure of -tubulin.

Figure 5.13 The tubulin dimer.

Microtubule Assembly
Protofilaments form by the end to end
association of tubulin
Protofilaments associate side by side in a
curved sheet which forms a hollow tube of
13 protofilaments
Microtubule extends by the addition of
tubulin dimers to both ends
The (+) or end grows fastest than the (-)
or end

Figure 5.16 Microtubules in a dividing cell.

Keratin
An intermediate filament
Structural proteins
Soft keratins help define the internal body
structure
Hard keratins- found in skin, hair, nails and
claws

Keratin
A coiled coil
A dimer of helices that wrap around each
other
Each polypeptide is composed of 7-residue
repeats with nonpolar residues in positions 1
and 4 of the repeat
As the dimer coils the nonpolar residues
contact and form a helix with a left hand
twist

Keratin is an intermediate
filament.
Keratin forms a
coiled-coil structure
shown in the three
representations
here.

Backbone

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Stick

Spacefilling

Intermediate Filaments
Dimers associate to form tetramers which
form octamers
The final filament is composed of 16 to 32
chains
Disulfide bonds can form between Cys
residues of neighboring chains
Keratin can stretch and bend
Keratin in dead structures such as hair can
stay intact for years

Figure 5.23 Model of an intermediate filament.

Collagen
Major extracellular protein
Major component of the extracellular
matrix
Nineteen known collagens
Collagen in bones and tendons best studied
A trimer of polypeptides with an unusual
composition

Collagen
In the mature protein, every third residue is
Gly
30% of remaining residues are Pro or Hyp
GlyProHyp is the most common repeat
Forms a narrow left-handed helix
Three of these wind into a right-handed
triple helix

Nonstandard Amino Acids

Found in Collagen
Hydroxyproline (Hyp)- proline with a OH
at the C4 position
Some 3-hydroxyproline in some types of
collagen
5-Hydroxylysine (Hyl)- lysine with a OH
in the C5 or position
Prolyl hydroxylase and lysyl hydroxylase
carry out the modifications

Collagen has a noteworthy

sequence.

Every 3rd amino acid = Gly

~30% of remaining amino acids are
proline or hydroxyproline.

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Collagen is
covalently
cross- Oxidation
linked.
Cross-linking
stabilizes
collagens
structure.

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Oxidation

Problem 5.27

Motor Proteins
An assortment of motor protein
Associated with microtubules and actin
filaments
Generate movement
Reorganization of cellular contents
Change cellular shape
Allow cell to swim or crawl

Myosin

A motor protein
Many types, found in nearly all cells
Muscle myosin is about 540 kD
Two large polypeptides that form two large heads
connected to a long tail-coil of the two coiled
chains
Each head has two binding sites
Actin binding site
ATP binding site

Myosin has two heads and a

long tail.

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Myosin binds to
ATP.

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Myosin-Actin Interaction
Numerous myosin molecules associate with
the tails interacting and the heads sticking
out- thick filaments
Actin and actin-binding proteins form thin
filaments
Myosin heads act as cross-bridges to the
actin filaments

ATP hydrolysis drives the physical

movement of myosin along an actin
filament.

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Myosin-Actin Reaction Cycle

The conversion of ATP to ADP causes
conformational changes in the head of
myosin
These changes are communicated to the
neck region and the actin binding site
These conformational changes lead to the
movement of myosin along the actin
filament

Kinesin
One of several motor proteins associated
with microtubules
MW ~ 380 kD
Two large globular heads with a coiled-coil
tail that has two light chain attached
Heads have an 8-strand sheet flanked by 3
helices
Each head has a tubulin binding site and a
ATP binding site

Kinesin is a microtubuleassociated protein.

Vesicle (cargo)
binding region

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Figure 5.17b Structure of kinesin.

Kinesin Transport
Cargo in the form of a vesicle is moved
along the microtubules
Vesicles are carried on the light chains of the
kinesin tail
Kinesin walks along the microtubule from
the (-) end toward the (+) end carrying its
cargo
Uses ATP to bring about conformational
changes so that chemical energy is
converted to mechanical energy

Kinesin transports cargo by moving

processively along a microtubule track.

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Kinesin transports cargo by moving

processively along a microtubule track .

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Kinesin transports cargo by moving

processively along a microtubule track.

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Kinesin transports cargo by

moving processively along a
microtubule track.

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Kinesin is a processive motor

Many cycles occur before the kinesin
dissociates from microtubules
Allows bulky cargo to be transported long
distances without being lost
Only moves in one direction