In the name of GOD

mistaureolinΜηχανική

27 Οκτ 2013 (πριν από 3 χρόνια και 7 μήνες)

127 εμφανίσεις

1

In the name of
GOD

2

Zeinab Mokhtari

10
-
Feb
-
2010

3

Isothermal titration calorimetry (ITC)

thermodynamics

of macromolecular
interactions in
biological

systems

direct measurement of heat exchange

extent of binding

a single experiment : binding constant, Gibbs free energy, enthalpy and entropy

Advantages

can be performed in a physiologically relevant buffer

the interacting species do not require immobilisation or chemical modification

an accurate determination of the stoichiometry (
independent of the binding affinity
)

Introduction

R=
1.98
cal mol
-
1

K
-
1

from the titration equivalence point

4

:

multiple binding events
analysis of systems involving

complexes

protein
formation of multi

binding of multivalent ligands

ligand

cooperativity

ITC for dissecting the thermodynamics of cooperativity

5

Selection of an Appropriate Binding Site Model

At the
beginning

of the experiment the calorimetric cell is
filled

with the
macromolecule
.

correction for displacement of liquid :

sensed calorimetrically

Normal

titration

6

nonlinear least squares curve fitting

necessary energy to maintain a constant temperature


(in microcalories per second)

each injection

binding

heat exchange

interaction

heat after each injection : the area under each peak

Heat
α

amount of binding

Saturation of macromolecule with the ligand

classical sigmoidal curve

decrease of the peaks magnitude until the peak
size reflects dilution and mechanical effects

7

model :

simplest
The

a single independent binding site

1
:
1
ligand/macromolecule

Wiseman isotherm

It is only in
c
value ranges of
approximately
1
to
1000
that
isotherms can be accurately
deconvoluted.

nonlinear least squares curve fitting

n
,
K
b

and ∆
H

8

Complex macromolecular interactions that display cooperativity

alternative binding models :


and the possible

multiple binding sites
between the binding sites

cooperativity

multiple

titration experiments :

The
contents

of the syringe and calorimetric cell are varied.

A
single

ITC experiment is often
insufficient

to sample
the shape of the binding isotherm and may not allow
derivation of the binding and cooperativity
parameters
.

9

Multiple Binding Sites

n
multiple ligand binding sites

two

different association constants:

the overall behaviour of the
n
sites (model dependent)

how binding occurs at each site (
model

dependent)

macroscopic

microscopic

1
:
1
interaction

only
one

association constant

determined by ITC

Overall binding constant

Stepwise binding constant

10

j

:any integer between
0
and
n

for ligation of the
j
th site:

ν :
the average number of ligand molecules bound per macromolecule


For multivalent macromolecules


11

Cooperativity

positive (synergistic)

negative (interfering)

α : a unitless term defined as the cooperativity constant

(
α <
1
)

(
α >
1
)

Noncooperative
(additive)

(
α =
1
)

Figure
1
.

Reaction scheme for the binding of heterogeneous ligands,
X
and
Y
, to a macromolecule,
M
, containing two binding sites.

12

binding sites

two
for a macromolecule with just

possible binding mechanisms

six
at least

The binding sites may be :


Identical

Independent (Neutral cooperativity (
α =
1
))

Negative cooperativity (
α <
1
)

Positive cooperativity (
α >
1
)


nonidentical

Independent (Neutral cooperativity (
α =
1
))

Negative cooperativity (
α <
1
)

Positive cooperativity (
α >
1
)

13

Cooperativity: Thermodynamics and Conformational Changes

conformational changes in macromolecular structure

large conformation changes

subtle changes

reinforcement of the interactions between the ligand and the receptor

Enthalpy


functional group interactions (ionic, hydrogen bonds, van der Waals interactions)

conformational changes

polarisation of the interacting groups

electrostatic complementarity

Entropy

a measure of disorder in a system


caused by changes in internal

loss of motion
Changes in the binding entropy reflect
of the molecules.
vibrations
and

rotations

upon complex formation

counterions
and the release of

Desolvation

entropy
-
enthalpy compensation

enthalpic chelate effect

structural tightening

classical entropic chelate effect

14

binding sites

dependent

two
binding to a macromolecule with

heterotropic

Gibbs

Helmholtz relationship

Vel
´
azquez
-
Campoy

15

entropy
: governed by

Type I

enthalpy
and

entropy
: governed by both

type II

enthalpic
: predominantly

type III

driven.
-
entropy
and

-
enthalpy
can be both

cooperativity

Positive

entropic cost of the

combined
cooperativity occurs when the

positive

Entropydriven
sequential binding events is
lower

than the
summation

of two independent events.

cooperative occurs when binding of the first ligand results

positive

driven
-
Enthalpy
in a
conformational

change

at the second binding site, rendering it
higher affinity

to
the ligand.

driven and occur when binding results in
-
entropy
cooperativity : mainly

Negative
a
loss of configurational entropy.

cooperativity : ligand binding leads to a conformational

negative
driven
-
Enthalpy

change that results in the
dissociation of a complex
.

homodimeric enzyme glycerol
-
3
-
phosphate:CTP transferase

multivalent carbohydrates to legume lectins

dissociation of the trimeric G
-
protein

cooperativity

Negative

three types of cooperativity


16

Reverse ITC Experiments

In order to fully resolve the binding and cooperative thermodynamics

to check the
stoichiometry

and the suitability of the binding
model

1
:
1

biomolecular reactions :

It is expected that the measured thermodynamic parameters are
invariant

when changing the
orientation

of the experiment.

BUT

One species may display greater aggregation when
concentrated
.

If normal and reverse titrations are insufficient to
fully describe the microscopic binding constants

Combination of the ITC data with other biophysical data that can
explore cooperativity, such as NMR and spectrofluorometry.

global fitting analysis

17

(
1
)

Selection of an appropriate model/binding polynomial


(
2
)

Calculation of the total macromolecule and ligand concentrations for each injections


(
3
)

Solvation of the the ligand conservation equation for each experimental point assuming
certain values


(
4
)

Calculation of the concentrations of each different complex or bound state


(
5
)
Calculation of the expected signal, assuming certain values for the binding enthalpies


(
6
)

Obtaining the optimal constants and enthalpies that reproduce the experimental data
using an iterative method,
i.e.
,
nonlinear least squares regression

General Analysis Procedure

mathematical methods for analysing cooperative ITC data

18

Analysis of Cooperativity Using the Binding Polynomial

Freire
et al.

major advantage : model independent

equilibrium conditions : the binding of ligand by a multivalent macromolecule may
be described by a
binding

polynomial

The number of binding sites should be known or fixed prior to analysis.

macroscopic association constants
and

enthalpy values

model specific constants

determining the correct model

starting point for data analysis in the absence of a validated binding model

19

partition function,
P
, of the system

summation of the different
concentrations of bound
species relative to the free
macromolecule concentration,


or

summation of the
concentration of free ligand in
terms of the macroscopic
association constant

fraction or population of each species

average number of ligand molecules bound per macromolecule

average excess molar enthalpy

average Gibbs free energy

model independent

20

three

states for a macromolecule with
two

binding sites

identical

binding polynomial for each
model

: summation of the terms in each
column

relative concentration of the
states

independent

general binding polynomial

21

ligand

homotropic
binding sites for a

two
a macromolecule with

example

Occupancy of the
binding sites

ligand concentration

association constants

cooperativity factor

event

heat
determined
-
nonlinear least squares regression analysis of the experimentally

22

accurate values for
β
j

and ∆
H
j

thermodynamic parameters

cooperativity factor

The value of the cooperativity parameter provides a very strong indication of the true binding model.

relate the macroscopic binding parameters to the microscopic binding parameters

23

Heterotropic Interactions

two different ligands

(
1
)

both ligands bind to the same binding site


(
2
)

both ligands bind to sites very close to one another, so that the ligands
themselves or binding site residues in the macromolecule interact


(
3
)

both ligands bind to binding sites distant to one another,
but are coupled
through a change in protein dynamics/conformation

Three mechanisms of
cooperativity

24

titration of ligand
X

into a calorimetric cell containing both
macromolecule, [
M
]
t

and ligand [
Y

]
t

assuming values of the
association

and
cooperativity constants

free concentrations of the reactants, [
M
], [
X
] and [
Y
]

concentrations of the complexes, [
MX
], [
MY
] and [
MXY
]

mass action law

solving the set of nonlinear equations numerically by the
Newton
-
Rhapson method

:

25

Only
one titration

experiment is required to determine the
interaction parameters instead of a series of experiments,
saving both
time

and
material
.

evaluating the heat effect,
q
i
,

associated with each
injection

by

nonlinear least squares fitting

26

Figure
2
.

Global and cooperative thermodynamic parameters associated
with the negatively cooperative binding of Fd to FNR
-
NADP+.

strong negative cooperativity

unfavourable
entropy

favourable
enthalpy

NADP+ complex with Fd
-
Titrations of FNR

α
=
0.17

Binding affinity is reduced by sixfold
when NADP+ is prebound to FNR.

27

Cooperativity of Long
-
Chain Macromolecules with Multiple Binding Sites

one
-
dimensional lattices

nucleic acids and carbohydrates

:
footprint

the minimal number of repeating units necessary to support binding (
l
)

characterisation of protein
-
lattice systems

affinity of the interaction

how the affinity varies with lattice heterogeneity

the binding site size (
l
)

whether ligand binding is cooperative

potential binding site overlap

cooperativity between neighbouring ligands

Ligands can bind to
lattices in three ways:

isolated binding

singly contiguous binding

doubly contiguous binding

intrinsic association constant

in the presence of neighbouring ligands

cooperativity factor,
α

28

homogenous lattice

actual number of free ligand binding sites on an unoccupied lattice

N
-

l
+
1

Figure
3
.

The three distinguishable types of ligand binding sites

29

Scatchard plot

a linear representation to facilitate data analysis

only linear when
l
=
1
and the binding sites are equivalent and independent

McGhee and von Hippel

any size of ligand footprint


cooperative interactions between contiguously bound ligands

infinite polymer

Extended by Tosodikov
et al. :
finite lattices

When
l >
1
, positive curvature reflecting the entropic resistance to saturation

30

Introduction of a cooperativity factor,
α

Scatchard plot is affected both by the entropic resistance
to saturation and the cooperativity parameter,
α
.

due to the entropic

negative cooperativity
Only linear if the apparent
.
positive cooperativity
resistance to saturation is compensated by real

the enthalpy associated with
the interaction between two
adjacent bound ligands

31

32

nature of the cooperativity

Change of the binding modes during the course of the titration

Non
-
cooperative system

Isolated ligands will bind initially, followed by singly contiguous ligands
and finally doubly contiguous ligands with two nearest neighbours.

Positively cooperative system

The ligands will immediately cluster forming doubly contiguous ligands.

Negatively cooperative system

Isolated ligands would form initially, and only when ligand accumulated
would singly contiguous ligands be observed. Ligands with two
neighbours would only accumulate at very high ligand concentration.

Reverse titrations

to fully characterise the binding isotherms

the roles of the ligand and macromolecule reversed

33

An alternative method of implementing the

Non
-
cooperative McGhee

von Hippel model

Shriver

change in concentration of bound protein as the result of the
i
th injection

the heat of
dilution

observed with each injection after
saturation

of the binding sites at the
end of titration

can be solved for values of
k
and
l

34

Example:

Chromatin Protein Sac
7
d Binding to DNA

Binds
non
-
cooperatively

and
non
-
specifically

to the
minor groove

of duplex DNA

induces a significant
kink

(
66
°
) in the DNA structure

Titrations :

Sac
7
d

and
poly(dGdC)

non
-
cooperative McGhee

von Hippel model

ITC data

25
°
C

moderate intrinsic affinity (approximately
833
nM)

ligand footprint of
4.3
base pairs


entropy driven (
17.5
kcal mol
¡
1
)

unfavourable enthalpic contribution (
9.2
kcal mol
¡
1
)

polyelectrolyte
effect

energy needed to

distort DNA

and

unwinding
,
pair unstacking
-
base
is associated with

Kinking
as well the

widening of the minor groove
, which leads to
bending
release of water and counterions

(which would contribute to the
.
backbone charge redistribution
term) due to

entropy
favourable

35

Global Analysis

multiple titrations, such as normal and reverse titrations

increase the information and precision of the parameters as
long as unrecognised systematic errors are not introduced

temperature and pH dependence

floating parameter
n

:

Reaction stoichiometry and concentration errors

n
is often a non
-
integer

In global analysis
only integral values

reflecting the
stoichiometry

are permitted.
Therefore it is necessary to accurately determine the stoichiometry prior to
global analysis by
alternative biophysical techniques
.

multisite and cooperative binding

SEDPHAT

Houtman
et al.

global analysis of data from a variety of biophysical techniques

36

Figure
4
.

Thermodynamics of the binding event determined by application of the
non
-
cooperative McGhee

von Hippel model to ITC data

Favourable entropy

Unfavourable

enthalpy

energetic penalty of kinking DNA

37

Global analysis of ITC data

role of
cooperativity

in the assembly of a threecomponent multiprotein complex

Example:

LAT, Grb
2
and Sos
1
Ternary Complex Assembly

To reduce the complexity :

LAT
pY
191
can bind one Grb
2
molecule, which in turn can bind one Sos
1
NT molecule.

Two titrations were performed:

LAT
pY
191

into Grb
2
alone

and
LAT
pY
191

into a stoichiometrically mixed
1
:
1
Grb
2
-
Sos
1
NT solution

global model for the
ternary interaction

K
d

=
286
nM



G
=
-
8
.
9
kcal mol
-
1



H
=
-
3
.
9
kcal mol
-
1

In the presence
of Sos
1
NT:

α =
0.54



g
=
0.37
kcal mol
-
1



h
=
-
3
.
9
kcal mol
-
1

A model without permitting cooperativity was unable to account for systematic difference
in the initial heats of injection for LAT phosphopeptide to Grb
2
in the presence and
absence of Sos
1
NT and resulted in an almost threefold increase in the
χ
2
of the fit.

38

Combination of ITC and NMR to Study Cooperativity

NMR

spectroscopy

measuring the occupancies of individual binding sites

determining the
microscopic binding affinities

site
-
specific data

macroscopic binding data from ITC

Full characterisation of the microscopic and macroscopic binding affinities

(
2
D HSQC)

isotope
-
enriched two
-
dimensional heteronuclear single
-
quantum coherence experiment

(A method of determining
cooperativity

using
NMR

spectroscopy)


isotopically
labeled

ligands
(usually
1
H and
13
C or
15
N)

unenriched macromolecule


Isotherms are generated by plotting the peak volume integration against molar ratio.


site
-
specific binding models

39

Example:

Glycocholate Binding to I
-
BABP

Human ileal bile acid binding protein (
I
-
BABP
)

glycocholate
binding sites for

two

the physiologically most abundant bile salt

intrinsically
weak

affinity

extremely strong
positive

cooperativity

But

low ligand

protein ratios : a significant amount of glycocholate remains unbound

high ligand

protein ratios : more ligand is bound

ITC and heteronuclear
2
D HSQC NMR

sequential model

glycocholate was isotopically labeled

three

main resonance peaks :

unbound

glycocholate, glycocholate bound at
site
1

and glycocholate bound at
site
2

site
-
specific binding model

NMR : microscopic affinities , cooperativity constant

multiple sites

different ligands

40

Thanks

Photo luminescence in coral