Transcript Document

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
The peptide backbone conformation can be described
In terms of two dihedral angles, Phi (F) and Psi (Y).
Phi is the dihedral angle for the N-C bond (hetero)
Psi is the dihedral angle for the C-C bond (same)
Dihedral angle
Berg Fig. 3.27
9/14/05
Trans and cis peptide bonds
The trans configuration is adopted
for almost all peptide bonds.
The peptide bond constrains the polypeptide
So in addition to the
peptide bond,
sterics inhibit
peptide
backbone
motion.
This comes
from peptide
backbone and
from R group.
Shown here,
F = 0,
Y=0
combination is
forbidden.
A few amino
acids have
unique sterics-
The peptide bond constrains the polypeptide
Ramachandran plot for
L-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
Glycine is highly flexible
A Ramachandran Plot for Polyglycine
“Conformational Space" constrained by peptide bond only
Fully allowed
At limits of
allowability
Pro
Conformational space has defined regions
Ramachandran plot for
L-Ala.
Region for righthanded a-helix.
"alpha"
The -helix
Ball-andStick models
The -helix
• 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 a-helix
The -helix
"wheel" depiction down helix axis
Helix dipole results
from orientation of
CO - NH hydrogen
bonds.
3
7
4
What does this
imply for the
preferred placement
of + and - amino
acids along a helix?
6
2
8
9
5
1
Conformational space has defined regions
Ramachandran plot for
L-Ala.
Region for -strands
beta
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
Antiparallel
Note:
•
H-bonds
•
R-groups
orientation,
distance.
•
Pleated character
•
Ave. strand length
is about 6 aa’s
0.7 nm
The -sheet
Parallel
Note:
•H-bonds not at
optimal angle.
•R-group orientations,
distances
•Pleated character
•Avg. Strand length ~
6 aa.
0.65 nm
The -turn
• 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.
Fibrous proteins
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
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
conformation
change
Carrier
conformation
change
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
Carrier
conformation
change
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.
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 prosthetic
group is sequestered in a deep heme binding pocket.
Heme
Fe2+
X
Fe3+
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 blood
exposed to the air is red.
Myoglobin binds oxygen
in muscle cells
Hemoglobin transports
oxygen in the blood
Hemoglobin subunits are structurally
similar 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 by
the distal histidine in myoglobin.
Binding of ligands
to 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
Distal His
Proximal His
Hb oxygen transport
Oxygen transport
•
Haemoglobin - oxygen transporter
Molecular mechanism of oxygen-binding
Role of DPG
Emphysema
Foetal vs Adult haemoglobin
His F8
His F8
His F8
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
Molecular mechanism of Hb co-operativity
Details
The tense state is stabilised by intersubunit interactions, in particular a network of
electrostatic interactions between amino acids.
2
1
2
1
Molecular mechanism of Hb co-operativity
The tense state is also stabilised by the binding of the
negatively charged co-factor, DPG.
DPG
Molecular mechanism of Hb co-operativity
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.
Hb without DPG
Y
Hb

Molecular mechanism of Hb co-operativity
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.
Molecular mechanism of Hb co-operativity
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 F8
His F8
His F8
Molecular mechanism of Hb co-operativity
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.
T state (lower affintiy for ligand)
R state (higher affinity for ligand)
Molecular mechanism of Hb co-operativity
The global conformational change is quite large: one  dimer
rotates relative to the other by 15° and shifts by 0.8 Å.
T-state
R-state
Top View
Molecular mechanism of Hb co-operativity
T-state
Molecular mechanism of Hb co-operativity
R-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.
Molecular mechanism of Hb co-operativity
The conformational
changes, initiated by
2 movement of helix F,
also rupture the
1 network of electrostatic
interactions between
subunits (across 
2 and interfaces).
This helps to relax the
1 contacts between
subunits.
Molecular mechanism of Hb cooperativity
Switch: Stabilisation of the R-state
Molecular mechanism of Hb cooperativity
High O2
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.
T
R
Low O2
Effect of CO2
CO2 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 Tstate, promoting deoxygenation.
The Bohr Effect
The 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.
Quick Time™ a nd a TIFF (Un co mp res sed ) d ec omp re sso r a re n ee ded to see th is p ictu re.
Christian Bohr’s Son
The Bohr Effect
The Bohr effect increases
the transport efficiency of
Hb.
66%
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.
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.
Example 1 Emphysema
People suffering from
emphysema (destructive
lung disease due to overactive 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
Y
Hb
1.0
0.5
Increasing
DPG
0.026
Y0
Ycomp
0.13
lungs
tissue
Oxyg en partial pressure (atm)
The role of 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.
This allows more
effective oxygen
transport (Ycomp > Y0)
because of increased
dissociation in the tissues
(at very low partial
pressures).
Y
Hb
1.0
0.5
Increasing
DPG
0.026
Y0
Ycomp
0.13
lungs
tissue
Oxyg en partial pressure (atm)
The role of DPG
Y
Hb
1.0
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.
0.5
Increasing
DPG
0.026
Y0
Ycomp
0.13
lungs
tissue
Oxyg en partial pressure (atm)
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 DPG
Example 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
Hb F
0.5
Hb A
Transfer to
foetus
Oxyg en partial pressure
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 Tstate and favouring the R-state.
The result is that oxygen affinity
goes up (since the R-state is a
higher affinity state).