Chapter 5 Protein Function Pratt3e Rev

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.

© 2014 John Wiley & Sons, Inc. All rights reserved.

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!© 2014 John Wiley & Sons, Inc. All rights reserved.

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 Identical in all Identical in Hb


Mb and Hb are similar in their secondary and tertiary

structures.

Myoglobin α-Subunit of Hemoglobinβ-Subunit of Hemoglobin

Heme

Even though myoglobin and hemoglobin have

only ~18% identical residues, their secondary and tertiary structures overlap almost

perfectly when superimposed!

Hb has quaternary structure, but Mb

does not.


Oxygen binds cooperatively to Hb.Dotted line represents

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 other subunits.© 2014 John Wiley &

Sons, Inc. All rights reserved.

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 TransportWhat is happening biochemically when you breathe?

From Metabolism

+ H2O


As pH , O2 affinity


From Metabolism

+ H2O

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

BPG decreases Hb’s O2 affinity.

Lower O2 affinity

Fra

ctio

nal

Sat

ura

tion

of

O2

BPG binds only to the tense (deoxy)

conformation of Hb.


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.

Actin monomer

Polymerization


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 equilbrium-

treadmilling• 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 site-

GTP

Figure 5.12 Structure of ß-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 acoiled-coil structureshown in the three

representationshere.

Backbone Stick Space-filling


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 δ 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.


Collagen is covalently

cross-linked.

Cross-linking stabilizes collagen’s structure.

Oxidation 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.


Myosin binds to ATP.


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.


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 microtubule-associated protein.

Vesicle (cargo) binding region


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.


Kinesin transports cargo by moving

processively along a microtubule track.


Kinesin transports cargo by moving processively along a microtubule track.


Kinesin transports cargo by moving processively along a

microtubule track.


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

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Chapter 5 Protein Function Pratt3e Rev