CONFORMATIONAL CHANGES IN PROTEIN STRUCTURE Structural changes arising from changes in state of...

CONFORMATIONAL CHANGES IN PROTEIN STRUCTURE

•Structural changes arising from changes in state of ligation

•Hinge motions in proteins

•Mechanisms of conformation changes (Haemoglobin, Serpins, muscle contraction)

•Higher level structural changes (GroELS)

Main chain conformation

The main chain conformation is the space curve that the backbone traces out

Under physiological conditions of solvent and temperature, all the molecules with the same amino acid sequence acquire the same native state

Protein architecture is the study of how folding patterns may be classified, internal interactions and determination of protein’s conformation by the amino acid sequence

The bond lengths and angles are fixed: the degrees of freedom of the chain involves four successive angles, in which the three are fixed and only the fourth can rotate around the bond linking the second and the theird atoms

The peptide bond constrains the polypeptide

Phi is the dihedral angle for the N-C bond (hetero)

Psi is the dihedral angle for the C-C bond (same)

The peptide backbone conformation can be describedIn terms of two dihedral angles, Phi (F) and Psi (Y).

Dihedral angle

9/14/05Berg Fig. 3.27

Trans and cis peptide bonds

The trans configuration is adopted for almost all peptide bonds.


Shown here,F = 0,Y = 0combination isforbidden.

So in addition to thepeptide bond, sterics inhibitpeptidebackbone motion.

This comes from peptidebackbone and from R group.

A few amino acids haveunique sterics-


Ramachandran plot forL-Ala.

"Allowed regions" of conformational space are in blue.

Two main allowed regions: = -57º; = -47º (R region)= -125º; = +125º ( region)

The mirror image of R is L and is only permitted for Gly.

R

A Ramachandran Plot for Polyglycine “Conformational Space" constrained by peptide bond only

Fully allowed

At limits ofallowability

Pro

Glycine is highly flexible

Conformational space has defined regions

Ramachandran plot forL-Ala.

"alpha"Region for right-handed a-helix.

The -helix

Ball-and-Stick models

• Most common type of 2˚ structural element (about 25% of the amino acids in proteins are in this structure)

• Right-handed helix• R-groups project outward, and provide the main

constraints on helical structure• Stability is greatly enhanced by internal van der

Waals contacts• H-bonds are in-line, optimum distance

The -helix

The a-helix

The -helix

Helix dipole resultsfrom orientation ofCO - NH hydrogen bonds.

What does this imply for the preferred placementof + and - amino acids along a helix?

12

3

4

5

6

9

7

8

"wheel" depiction down helix axis

Conformational space has defined regions

Ramachandran plot forL-Ala. beta

Region for -strands

The -strand

•Highly extended form of polypeptide chain.•3.5 Å between adjacent residues (1.5 Å for a-helix!)

•Adjacent side chains point in opposite directions.•A beta strand usually is associated with other beta strands….

The -sheet

Note:• H-bonds• R-groups

orientation, distance.

• Pleated character• Ave. strand length

is about 6 aa’s

Antiparallel

0.7 nm

The -sheet

Parallel

0.65 nm

Note: •H-bonds not at optimal angle.•R-group orientations, distances•Pleated character•Avg. Strand length ~ 6 aa.

• Connects other 2˚ structure elements, causes the backbone to reverse direction.

• a 4 amino acid loop, reversing direction 180o

• An H-bond between C=O of aa1 and NH of aa4 is found

• aa3 is often Gly, which is small, flexible• aa2 may be Pro, whose cis-conformation

turns tightly• Usually occurs at surfaces, connecting

antiparallel b-strands• There are different types (I, II, III)

depending on conformation.

The -turn

In general, fibrous proteins are- built up from a single element of secondary structure- insoluble in water (lots of hydrophobic residues)- involved in structural roles within the cell

Fibrous proteins

Sidechain conformation

Sidechain conformation are described by angles of internal rotation (1 and 5)

Different sidechains have different degrees of freedom

Arg 5

Gly and Ala 0

Rotamers = conformations of any sidechains corresponding to different combinations of values

C 60º (g-)

180º (t)

60º (g+)

Rotamer libraries = statistical analysis of patterns of conformational angles in well-determined protein structures (collections of preferred sidechain conformations)

Local backbone conformations as well as secondary protein structures limit the possible range of sidechain internal rotations due to potential steric collisions

The small number of possible internal conformations for sidechains make rotamer libraries of sidechains ideal for modelling

Structural changes from changes in ligation

•Carrier proteins cycle between conformations in which a solute binding

site is accessible on one side of the membrane or the other.

•There may be an intermediate conformation in which a bound substrate is

inaccessible to either aqueous phase. With carrier proteins, there is never an

open channel all the way through the membrane.

conformation

change

conformation

change

Carrier mediated solute transport

Carrier proteins cycle between conformations in which a solute

binding site is accessible on one side of the membrane or the other.

There may be an intermediate conformation in which a bound

substrate is inaccessible to either aqueous phase. With carrier

proteins, there is never an open channel all the way through the

membrane.

Proteins as carrier

conformation

change

conformation

change

Carrier mediated solute transport

Conformational changes in proteins

In some proteins, the binding with ligands or other proteins can lead to dramatic conformational changes (Haemoglobin + Oxigen)

In some other cases changes in the structure are not significant or affect the protein only locally (Myoglobin + Oxigen)

Allosteric transitions involve long-range integrated conformational changes (haemoglobin)

Some other proteins act as pump or motors through changes in conformation (Myosin of muscle, ATPase, GroELS)

Myoglobin

Heme

3

Myoglobin binds oxygen in muscle cells. The heme prostheticgroup is sequestered in a deep heme binding pocket.

Heme

Fe2+

Fe3+

X

Heme function

Oxygen is not very soluble in water.To transport oxygen in our blood we use a protein, hemoglobin.None of the amino acids efficiently binds oxygen.Hemoglobin uses iron (Fe2+) to coordinate oxygen.Free iron generates oxygen radicals that are harmful.Hemoglobin sequesters iron in a heme prosthetic group.Fe2+ in a heme is less reactive. The nitrogens prevent conversion from

the ferrous state (Fe2+) to ferric state (Fe3+)In free heme molecules, binding of oxygen converts Fe2+ to Fe3+.Sequestering the heme in hemoglobin prevents this conversion.Binding of oxygen by hemoglobin changes the properties of heme.

The heme changes color, this is why veins are blue and bloodexposed to the air is red.

Myoglobin binds oxygenin muscle cells

Hemoglobin transportsoxygen in the blood

Hemoglobin subunits are structurallysimilar to myoglobin

Specificitiy of ligand binding

Heme binds carbon monoxide (CO) 20,000 times better than O2

Myoglobin binds CO 200 times better than O2

The protein portion of myoglobin sterically interferes with CO binding to the heme.

A histidine (the distal His) interacts with the ligand (CO or O2).

Oxygen binds to heme with the O2 axis at an angle.This binding conformation is accommodated by myoglobin.

Carbon monoxide normally binds to heme with the CO axis perpendicular to the plane of the porphyrin ring.

This binding conformation is sterically hindered bythe distal histidine in myoglobin.

Proximal His

Distal HisBinding of ligandsto the heme of

myoglobin

Even though the difference between deoxy and oxy forms of myoglobine are not huge, the oxigen-binding site is blocked and the molecule must open up during the process

of capture and oxygen release

Oxygen transport

• Haemoglobin - oxygen transporter

Molecular mechanism of oxygen-binding

Role of DPG

Emphysema

Foetal vs Adult haemoglobin

Oxygen transport

• Haemoglobin - oxygen transporter

Molecular mechanism of oxygen-binding

Role of DPG

Emphysema

Foetal vs Adult haemoglobin

His F8

His F8 His F8

Hb oxygen transport

Max Perutz (1914-2002)

Molecular mechanism of Hb co-operativity

Overview

In the low-affinity, tense (T) state, deoxy-Hb (no bound oxygen) contains numerous intersubunit interactions. DPG is bound on one face of the tetramer, at the subunit interface

Upon oxygen binding, the architecture of the Hb tetramer transmits conformational changes from the oxygen binding site to the intersubunit interfaces (and vice versa). The DPG binding site is altered and DPG dissociates from the Hb molecule.

As oxygen binds, the intersubunit contacts loosen; each subunit becomes more like myoglobin and adopts Mb-like affinity for oxygen


2

1

1

2

Details

The tense state is stabilised by intersubunit interactions, in particular a network of electrostatic interactions between amino acids.


The tense state is also stabilised by the binding of the negatively charged co-factor, DPG.

DPG


DPG plays a major role in T-state stabilisation; in the absence of DPG, Hb switches to the R-state (high affinity) and is not capable of displaying co-operativity.

Y

Hb without DPG

Hb


Oxygen binding to heme causes the Fe atom to move about 0.4 Å (0.04 nm) into the plane of the heme

This displaces the proximal histidine (His F8) and helix F to which it is attached.


As well as displacing helix F, the flattening of the porphyrin ring upon oxygen binding causes displacement of a Val residue in the turn between helix F and helix G.

His F8His F8 His F8


T state (lower affintiy for ligand)

R state (higher affinity for ligand)

The F helix is also in close proximity to the inter-subunit interfaces. Thus, oxygen binding in one subunit is communicated to the other subunits.

Ultimately the binding of oxygen produces a large conformational change.


The global conformational change is quite large: one dimer rotates relative to the other by 15° and shifts by 0.8 Å.

Top View

T-state R-state

Molecular mechanism of Hb co-operativityT-state


In the R-state, there is a looser association of subunits.

Once converted to the R-state, the barrier to oxygen binding is removed and oxygen affinity rises.

The last oxygen molecule to bind binds 20-300 times tighter than the first.

R-state


The conformational changes, initiated by movement of helix F, also rupture the network of electrostatic interactions between subunits (across and interfaces).

This helps to relax the contacts between subunits.

2

1

1

2


Switch: Stabilisation of the R-state


Effectively, the binding energy of the first oxygen molecule is partly “consumed” in order to relax the Hb structure (loosen the intersubunit contacts); thus the initial oxygen affinity is low.

Once converted to the R-state, the barrier to oxygen binding is removed, thereby increasing oxygen affinity.

Low O2

High O2

T R

Effect of CO2CO2 and O2 antagonise each other’s binding

The ability of Hb to sense and respond appropriately to differing oxygen levels is a well-balanced mechanism for self regulation. Hb is also sensitive to pH (H+ ion concentrations) and to carbon dioxide (CO2) levels. In very active cells, such as contracting muscle, CO2 and H+ concentrations increase.

CO2 can bind to Hb and in doing so stabilises the low-affinity T-state.

CO2 can react with the amino termini of polypeptides to form a negatively charged carbamate. This modification at the N-terminal of the -subunits generates an electrostatic interaction with Arg 141 from the nearest -subunit which stabilises the T-state, promoting deoxygenation.

The Bohr EffectThe Bohr effect

H+ can also bind to Hb to stabilise the low-affinity T-state.

At low pH (high [H+]), His 146 from the -subunits becomes protonated (positively charged) and can make a salt-bridge to Asp 94 from the same chain. This helps to stabilise the interaction between the COOH of His 146 and Lys 20 from the nearby -chain

In active cells therefore, Hb even more readily gives up its bound oxygen; thus oxygen is automatically targeted to the cells where it is most needed. This is known as the Bohr effect.

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

Christian Bohr’s Son

The Bohr Effect

The Bohr effect increases the transport efficiency of Hb.

Hb also functions to assist the removal of CO2 from the bloodstream. In the lungs the Bohr effect works in reverse to facilitate the dissociation of CO2. Since CO2 and O2 bind antagonistically, the presence of high concentrations of O2 in the lungs, induces release of bound CO2 from Hb molecules arriving there from the tissues.

66%

4.7 The role of 2,3-diphosphoglycerate (DPG)

The body can regulate DPG levels and the affinity of Hb for this co-factor to control the oxygen affinity of Hb in response to particular circumstances.

Y

1.0

0.5

0.130.026

Oxygen partial pressure (atm)

tissue lungs

Y0

Hb

IncreasingDPG Ycomp

Example 1 - Emphysema

People suffering from emphysema (destructive lung disease due to over-active neutrophil elastase) have a lower partial pressure of oxygen in their lungs.

Their bodies compensate by increasing DPG levels. This increases the stability of the T-state and lowers the affinity for oxygen at the very low oxygen partial pressures which are found in the tissues of the body

The role of DPG

Y

1.0

0.5

0.130.026


tissue lungs

Y0

Hb

IncreasingDPG Ycomp

This allows more effective oxygen transport (Ycomp > Y0) because of increased dissociation in the tissues (at very low partial pressures).

The body can regulate DPG levels and the affinity of Hb for this co-factor to control the oxygen affinity of Hb in response to particular circumstances.

The role of DPG

Serious mountain climbers need to spend time acclimatising before ascending too high. This gives the body time to increase DPG levels to allow the climber to cope better with the lower oxygen pressures that occur at high altitudes.

Y

1.0

0.5

0.130.026


tissue lungs

Y0

Hb

IncreasingDPG Ycomp

The role of DPG

Smoking makes emphysema worse if you have a mutant version of 1-antitrypsin (=anti-elastase). The smoke oxidises Met 358 in the inhibitor molecule, a residue essential for its binding to elastase.

1-antitrypsin

The role of DPGExample 2 - Foetal haemoglobin

The foetal oxygen transport problem is quite different to that of children or adults.

Foetal haemoglobin must transport oxygen from the mother’s placenta to itself; it has to compete for oxygen with the maternal haemoglobin.

The foetus competes effectively by making a Hb molecule (Hb F) with higher oxygen affinity, especially at lower partial pressures, than adult Hb (Hb A) .

Y

1.0

0.5

Oxygen partial pressure

Hb F

Hb A

Transfer tofoetus

The role of DPG

Hb F is made from 2 and 2 chains. chains are derived from a gene that is only active during the foetal stage of development. The gene for the chain is only switched on (and the gene switched off) after birth.

The role of DPG

and chains have a number of amino acid sequence differences. Most notably, His 143 in , at the DPG binding site, is replaced by Ser in the chain. The loss of a positively charged residue reduces the affinity of Hb F for DPG, thus destabilising the T-state and favouring the R-state. The result is that oxygen affinity goes up (since the R-state is a higher affinity state).

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