BIO439 2011 02 Thermodynamics

2.
Thermodynamics
2. Thermodynamics
2.1. Scope and definitions
2.2. Equilibrium constant, free energy and spontaneous reaction
2.3. Applications in biology
2.3.1. Transfer free energy and predictions of
transmembrane segments
2.3.2. Protein folding and stability
2.3.3. DNA stability and translation fidelity
2.4. Binding equation and binding curve
BIO429 LESSON 2 1
2.5. Measuring binding
2.5.1. Equilibrium dialysis
2.5.2. Filter binding assay
2.5.3. Gel shift assay
2.5.4. Enthalpy and entropy
2.5.5. Isothermal titration calorimetry (ITC)
2.5.6. Equilibrium and kinetics
2.5.7. Surface plasmon resonance (SPR)
Interactions are central to biology
Rgulation de la transcription
ARN polymrase
ADN polymrase
2 BIO429 LESSON 2
Interactions are central to biology
3 BIO429 LESSON 2
+
+
Thermodynamics Binding conformational changes
+
4 BIO429 LESSON 2
In physics, thermodynamics is the study of
- the conversion of heat energy into different forms of
energy (in particular, mechanical, chemical, and electrical
Thermodynamics
from the Greek - , therme, meaning heat
- , dynamis, meaning power
energy (in particular, mechanical, chemical, and electrical
energy)
- the conversion of different energies into heat energy
- its relation to macroscopic variables such as temperature,
pressure, and volume.
5 BIO429 LESSON 2
Systme
Environnement
Univers
Univers = Systme + environnement
Systme ouvert: Matire et nergie
Systme ferm: Energie
Systme isol: Rien
Matire
Energie
La thermodynamique permet de dcrire un niveau macroscopique la matire
et les changements physiques et chimiques quelle subit.
Cette description repose sur une reprsentation simplifie de la ralit (un
modle) et sur un nombre restreint de lois.
6 BIO429 LESSON 2
Equilibrium versus non-equilibrium thermodynamics
Non-equilibrium thermodynamics is a branch of thermodynamics concerned
with studying time-dependent thermodynamic systems, irreversible
transformations and open systems.
In contrast to most physical theories that rely on thermodynamic equilibrium,
most systems found in nature are not in equilibrium.
most systems found in nature are not in equilibrium.
Real systems are not isolated from their environment and are therefore
continuously sharing energy with other systems.
This sharing of energy includes being driven by external energy sources as
well as dissipating energy.
7 BIO429 LESSON 2
la grandeur la plus utile pour lanalyse des systmes biologiques
lquilibre est lnergie libre de GIBBS
Lapplication la plus simple de la thermodynamique aux systmes
biologiques et chimiques est ltude des phnomnes dquilibre
et
K ln RT G =
2.2. Equilibrium constant, free energy and
spontaneous reaction
K ln RT G =
+ = ln RT G G
8 BIO429 LESSON 2
Chemical potential
9 BIO429 LESSON 2
10 BIO429 LESSON 2
11 BIO429 LESSON 2
The free energy G is the key parameter, because its value under a particular
set of reactant concentrations dictates the direction of biomolecular equilibria.
0 G <
If its sign is negative, the binding
reaction or conformational
transition will proceed
spontaneously to an extent
governed by the magnitude of G.
G
i
b
b
s

f
r
e
e

e
n
e
r
g
y

c
o
n
t
e
n
t
,

G

(
k
J
)
7.5
8.0
8.5
Free energy and direction of the reaction
0 G >
0 G =
governed by the magnitude of G.
If its sign is positive, the
magnitude of G specifies the
energy needed to drive the
reaction to form product.
If it is equal to zero, the system
is at equilibrium
/K
0.001 0.01 0.1 1 10 100 1000
G
i
b
b
s

f
r
e
e

e
n
e
r
g
y

c
o
n
t
e
n
t
,

G

(
k
J
)
5.5
6.0
6.5
7.0
12 BIO429 LESSON 2
Octanol
(X )
(X
AA
)
eau
(X
AA
)
octanol
K
Transfert
=
Application de lanalyse thermodynamique lquilibre:
chelles dhydrophobicit et prdiction des protines membranaires
2.3.1. Transfer free energy and predictions of transmembrane segments
Eau
(X
AA
)
octanol
(X
AA
)
eau
G
0
Transfert
= - RT ln K
Transfert
13 BIO429 LESSON 2
Hydrophobicity scales
(Wimley, Creamer & White, 1996)
14 BIO429 LESSON 2
Hydrophobicity scales
(Wimley, Creamer & White, 1996)
15 BIO429 LESSON 2
Predicting transmembrane regions
16 BIO429 LESSON 2
Que signifie se replier (protein folding)
pour une protine?
17 BIO429 LESSON 2
Former des hlices , des brins et des coudes
Adopter un arrangement prcis de ces lments de structure
secondaire
Les chanes latrales sont compactes et imbriques les unes dans les
autres. Restriction de la mobilit des chanes latrales
Ejection de leau du cur de la protine et formation dun cur
Que signifie se replier pour une protine?
(protein folding)
Ejection de leau du cur de la protine et formation dun cur
hydrophobe
Toutes les molcules adoptent la mme structure avec un arrangement
prcis des chanes latrales (site actif)
A temprature ordinaire, une protine native nest entirement rigide
mais conserve une certaine flexibilit
Lexistence dune structure identique dans toutes les molcules et dune
certaines rigidit permet la dtermination de la structure
(cristallisation, RMN)
18 BIO429 LESSON 2
D
N
Comment une protine se replie-t-elle ?
19 BIO429 LESSON 2
LYS GLU THR ALA ALA ALA LYS PHE GLU ARG
GLN HIS MET ASP SER SER THR SER ALA ALA
SER SER SER ASN TYR CYS ASN GLN MET MET
LYS SER ARG ASN LEU THR LYS ASP ARG CYS
LYS PRO VAL ASN THR PHE VAL HIS GLU SER
LEU ALA ASP VAL GLN ALA VAL CYS SER GLN
LYS ASN VAL ALA CYS LYS ASN GLY GLN THR
ASN CYS TYR GLN SER TYR SER THR MET SER
ILE THR ASP CYS ARG GLU THR GLY SER SER
LYS TYR PRO ASN CYS ALA TYR LYS THR THR
GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS
Quest-ce qui contrle le repliement dune protine?
GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE
ASP ALA SER VAL
Anfinsen C. : Unfolding of a purified protein is fully reversible
(Anfinsen C., Science 1972, )
Protein folding is a spontaneous and autonomous process
20 BIO429 LESSON 2
Exprience de dnaturation de protine
21 BIO429 LESSON 2
Ribonuclease A
22 BIO429 LESSON 2
23 BIO429 LESSON 2
24 BIO429 LESSON 2
LYS GLU THR ALA ALA ALA LYS PHE GLU ARG
GLN HIS MET ASP SER SER THR SER ALA ALA
SER SER SER ASN TYR CYS ASN GLN MET MET
LYS SER ARG ASN LEU THR LYS ASP ARG CYS
LYS PRO VAL ASN THR PHE VAL HIS GLU SER
LEU ALA ASP VAL GLN ALA VAL CYS SER GLN
LYS ASN VAL ALA CYS LYS ASN GLY GLN THR
ASN CYS TYR GLN SER TYR SER THR MET SER
ILE THR ASP CYS ARG GLU THR GLY SER SER
LYS TYR PRO ASN CYS ALA TYR LYS THR THR
Protein folding is a spontaneous and autonomous process
GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE
ASP ALA SER VAL
Anfinsen C. : Unfolding of a purified protein is fully reversible
(Anfinsen C., Science 1972, )
Merrifield R.B. : A synthetic polypeptide chain folds to the
same structure than the natural protein
(Synthetic RNase A: 78 % activity, 0.4 mg was synthesized, 2.9 % overall yield, average
yield ~ 97% per coupling step. B. Gutte & R. B. Merrifield, J. Am. Chem. Soc. 1969,
91, 501-2)
25 BIO429 LESSON 2
D
N
Conformational change Protein folding
U
I
a
k
12
k
21
U
I
a
k
12
k
12
k
21
k
21
eq
k f
] D [
= = =
N
D
D
12
21
N
D
eq
eq
k
k
f
f
] N [
] D [
K = = =
K ln RT G =
26 BIO429 LESSON 2
Free energy
U
Thermodynamic stability of proteins
U
I
a
k
12
k
21
U
I
a
k
12
k
12
k
21
k
21
12
21
N
D
eq
eq
k
k
f
f
] N [
] D [
K = = =
N D
Folding reaction
N
G
fold
< 0
27 BIO429 LESSON 2
K ln RT G =
F
r
a
c
t
i
o
n

f
o
l
d
e
d
50
100
Folded
Unfolding transitions are highly
cooperative
[Denaturant]
0 1 2 3 4 5
F
r
a
c
t
i
o
n

f
o
l
d
e
d
0
50
Unfolded
28 BIO429 LESSON 2
29 BIO429 LESSON 2
Fraction de I (f
I
)
0.6
0.8
1.0
~ f
D
U
I
a
k
12
k
21
U
I
a
k
12
k
12
k
21
k
21
eq
k f
] D [
= = =
N D
Equilibrium measurements
f
o
l
d
e
d
[Urea] (M)
0 1 2 3 4 5 6
0.0
0.2
0.4
~ f
N
30 BIO429 LESSON 2
12
21
N
D
eq
eq
k
k
f
f
] N [
] D [
K = = =
K ln RT G =
F
r
a
c
t
i
o
n

f
o
l
d
e
d
G(cal mol
-1
)
1000
2000
3000
) O H ( G
2

m
Tanford (1970) Adv. Prot. Chem. 24, 2-95
Santoro & Bolen (1988) Biochemistry 27, 8063-8068
U
I
a
k
12
k
21
U
I
a
k
12
k
12
k
21
k
21
eq
k f
] D [
= = =
N D
[Urea] (M)
0 1 2 3 4 5 6
-3000
-2000
-1000
0
] Urea [ m ) O H ( G G If
2
=
m
31 BIO429 LESSON 2
12
21
N
D
eq
eq
k
k
f
f
] N [
] D [
K = = =
K ln RT G =
F
r
a
c
t
i
o
n

f
o
l
d
e
d
0.4
0.6
0.8
1.0
Circular dichroism at 222nm
Fluorescence at 320nm
[Urea] (M)
0 1 2 3 4 5 6 7
F
r
a
c
t
i
o
n

f
o
l
d
e
d
0.0
0.2
0.4
Jamin & Baldwin (1996) Nature Structural Biology 3, 613-618
32 BIO429 LESSON 2
33 BIO429 LESSON 2

Protine

DG (kJ/mol) (25C)

BPTI 44.2
CI2 27.7
Ubiquitine 40.1
RNAse T1 37.5
Marginal thermodynamic stability of proteins
RNAse T1 37.5
Cytochrome C 37.1
Barnase 48.9
RNAse A 27.0
Myoglobine 40.6

Interaction de faible
energie

Liaison covalente

~ 4-20

~ 400

34 BIO429 LESSON 2
DNA melting can be monitored by
absorbance at 260 nm
35 BIO429 LESSON 2
Thermal melting of DNA
36 BIO429 LESSON 2
37 BIO429 LESSON 2
Average = 4.54 kcal/mol
1.1 kcal/mol per bp
Average = 5.41 kcal/mol
1.1 kcal/mol per bp
38 BIO429 LESSON 2
K
K = [B] / [A]
G= - 3.3 kcal/mol K = exp(- G/RT) = 211
f
B
= [B] / ([A] + [B]) = ([B] / [A]) / (1 + ([B] / [A]))
fB = 211 / 212 = 0.995
Dans une population de 1000 molcules :
A B
5 A
995 B
39 BIO429 LESSON 2
Cognate, near-cognate and non-cognate codonanticodon pairing.
The anticodon must interact with the first two codon positions
according to the WatsonCrick base pairing rules, whereas some
deviation, e.g. the GU pair, is allowed at the third, or wobble
position.
40 BIO429 LESSON 2
GAG
UUC
x
K = [B] / [A]
f
B
= [B] / ([A] + [B]) = ([B] / [A]) / (1 + ([B] / [A]))
f
B
= 35 / 36 = 0.972
Dans une population de 1000 molcules :
28 A
972 B
41 BIO429 LESSON 2
Insights into the decoding mechanism from recent ribosome structures
During the decoding process, tRNA selection by the ribosome is far more
accurate than expected from codonanticodon pairing.
Recent crystal structures show that the specific recognition of base-
pairing geometry leads to a closure of the domains of the small subunit
around cognate tRNA. This domain closure is likely to trigger subsequent
steps in tRNA selection.
42 BIO429 LESSON 2
Accuracy of protein synthesis depends on:
specificity of the codonanticodon interaction
kinetic proofreading
direct involvement of the ribosome - conformational change of RNA 16s
43 BIO429 LESSON 2
video
44 BIO429 LESSON 2
Recognition of base pair structure
45 BIO429 LESSON 2
In order to assess the biological significance of an interaction we
need to have some idea how tightly the molecules bind, especially in
relation to their physiological concentrations .
The equilibrium dissociation constant, Kd, describes the strength of
binding.
How quickly the association or dissociation occurs - the kinetics of
How quickly the association or dissociation occurs - the kinetics of
binding - can also be very important. However, here we will talk about
kinetics only in the context of how kinetic and equilibrium constants
relate to one another.
46 BIO429 LESSON 2
Simple bimolecular association-dissociation reaction
M
(Macromolecule)
+
L
(Ligand)
ML
The tighter M and L bind to one another, the more ML there will be at
equilibrium.
Conversely, the weaker the binding of M to L, the more unbound M and
unbound L there will be at equilibrium.
(Macromolecule) (Ligand)
47 BIO429 LESSON 2
The equilibrium dissociation constant is defined as the product of the
concentrations of the unbound species divided by the concentration of
Simple bimolecular association-dissociation reaction
M
(Macromolecule)
+
L
(Ligand)
ML
The equilibrium dissociation constant is defined as the product of the
concentrations of the unbound species divided by the concentration of
the product.
In this case:
K
d
= 10
-12
M stronger binding than K
d
= 10
-9
M
] ML [
] L [ ] M [
K
d
=
48 BIO429 LESSON 2
Fraction of macromolecule bound : [ML]/[Mtotal]
If one plots the fraction of M bound vs the concentration of
unbound (free) ligand L, a characteristic hyperbolic binding curve
is obtained.
The shape of the binding curve
49 BIO429 LESSON 2
Binding equation - Simple case
Simplest case [L] >>> [M]
] ML [
] L [ ] M [
K
T
d
=
] ML [
=
Free T
] L [ ] L [ =
] ML [ ] M [
] ML [
+
=
T d
T
d
T
d
T
d
T
d
T
] L [ K
] L [
K
] L [
1
K
] L [
K
] L [
] M [ ] M [
K
] L [
] M [
+
=
+
=
+
=
50 BIO429 LESSON 2
In a standard equilibrium dialysis
assay you begin with two chambers
separated by a dialysis membrane.
The molecular weight cut off (MWCO)
of this membrane is chosen such that it
will retain the receptor component of
the sample (the element which will bind
the ligand).
the sample (the element which will bind
the ligand).
A known concentration and volume
of ligand is placed into one of the
chambers. The ligand is small enough to
pass freely through the membrane.
51 BIO429 LESSON 2
A known concentration of receptor
is then placed in the remaining
chamber in an equivalent volume
to that placed in the first chamber.
As the ligand diffuses across the
membrane some of it will bind to the
receptor and some will remain free in
solution. The higher the affinity of the
interaction, the higher the concentration
of ligand that will be bound at any time.
52 BIO429 LESSON 2
Diffusion of the ligand across the
membrane and binding of the ligand
continues until equilibrium has been
reached.
At equilibrium, the concentration of
ligand free in solution is the same in
both chambers.
In the receptor chamber, however, the
overall concentration is higher due to
the bound-ligand component.
The concentration of free ligand in the
ligand chamber can then be used
to determine the binding characteristics
of the samples.
53 BIO429 LESSON 2
54 BIO429 LESSON 2
Filter binding
Cro repressorDNA complex
55 BIO429 LESSON 2
56 BIO429 LESSON 2
57 BIO429 LESSON 2
58 BIO429 LESSON 2
59 BIO429 LESSON 2
Premire loi : Lnergie peut tre convertie dune forme dans
une autre mais ne peut tre ni cre ni dtruite
Deux lois fondamentales de la thermodynamique
E = q + W
Energie potentielle
Energie cintique
Les diffrentes formes dnergie peuvent se transformer les unes dans les autres
60 BIO429 LESSON 2
Seconde loi : Lentropie totale de lunivers (systme + environnement)
DOIT AUGMENTER dans tout processus spontan
Raction spontane
S
Total
= S
System
+ S
Surroundings
> 0
Effort organis ncessitant un apport dnergie
61 BIO429 LESSON 2
Equilibrium Thermodynamics is the systematic study of transformations of
matter and energy in systems as they approach equilibrium.
Equilibrium thermodynamics, in origins, derives from analysis of the Carnot
cycle
Here, typically a system, as cylinder of gas, is set out of balance via heat input
from a combustion reaction. Then, through a series of steps, as the system
settles into its final equilibrium state, work is extracted.
settles into its final equilibrium state, work is extracted.
Sadi Carnot
62 BIO429 LESSON 2
The free energy is a balance between enthalpy and entropy.
S T H G =
The enthalpy change reflects the amount of heat energy required
to achieve a particular state.
The entropy measures how easily that energy might be
distributed among various molecular energy levels.
= ln k S
B
63 BIO429 LESSON 2
K ln RT G =
+ = ln RT G G
S T H G =
|
|
\
|
=
) T / 1 (
K ln
R H
VH
GIBBS free energy
Enthalpy
p
p
T
H
C
|
\
|

=
( )
0 p 0 T T
T T C H H + =
|
|
\
|
+ =
0
p 0 T T
T
T
ln C S S
( )
(
|
|
\
|
+ + =
0
0 p 0 T 0 T
T
T
ln T T C S T H G
Heat capacity
64
BIO429 LESSON 2
65 BIO429 LESSON 2
Octanol
(X
AA
)
octanol
(X
AA
)
eau
Eau
66 BIO429 LESSON 2
67 BIO429 LESSON 2
68 BIO429 LESSON 2
Antoine-Laurent Lavoisier au travail
Huile sur toile par Jacques-Louis David
69 BIO429 LESSON 2
Two calorimetry methods
Differential Scanning Calorimetry Isothermal Titration Calorimetry
Two calorimetric methods, differential scanning calorimetry (DSC) and
isothermal scanning calorimetry (ITC), dominate in biophysics and biochemistry.
Differential Scanning Calorimetry
DSC
Isothermal Titration Calorimetry
ITC
Stabilit des molcules et des
complexes
Solvatations
Mesure de laffinit, de la
stoichiomtrie et du nombre de
site de fixation
70 BIO429 LESSON 2
Avantages
(1) Mesure la chaleur de raction en
solution. Pas besoin de marquage et
technique non-invasive.
(2) H est mesur directement. Pas
besoin de varier la temprature.
Limitations
Limitations
(1) Utilisation dune grande quantit de
matriel.
(2)Sensibilit intrinsque. Les valeurs
typiques de H pour la formation de
complexes sont de lordre de 10
kcal/mol et donc les mesures sont
limites aux systmes dont le Kd est
de lordre de 10-7 M ou plus petit.
71 BIO429 LESSON 2
Ligand
Lexprience de base
(1) Remplir la seringue avec le ligand
forte concentration
(2)Remplir le rservoir avec la protine
faible concentration
(3)Titrer la protine en ajoutant des
petites quantits de ligand. Aprs
Reference
cell
Sample
cell
Adiabatic jacket
(3)Titrer la protine en ajoutant des
petites quantits de ligand. Aprs
chaque addition, linstrument rajuste
la temprature de la cellule par rapport
la temprature de la cellule de
rfrence et mesure la chaleur
ncessaire pour raliser cet ajustement
(4)Soustraire le titrage blanc pour tenir
compte de la chaleur de dilution du
ligand
72 BIO429 LESSON 2
Rsultats de lexprience
Le rsultat brut est un graphe montrant la chaleur (mJ ou mcal)/sec
en fonction du temps
73 BIO429 LESSON 2
Les donnes doivent tre traites pour obtenir un isotherme de
fixation
Intgration de la surface sous chaque pic (fournit la chaleur
libre/absorbe pour chaque ajout de ligand) Q
Division par la concentration dilue de ligand ajout chaque point du
titrage Q / [X] (axe des y)
titrage Q / [X] (axe des y)
Calcul de la concentration de ligand ajout (axe des x)
74 BIO429 LESSON 2

Q

/

[
X
]
75 BIO429 LESSON 2
76 BIO429 LESSON 2
Isostructural Is Not Isoenergetic
77 BIO429 LESSON 2
A Thermodynamic Signature for DNA Binding Mode
78 BIO429 LESSON 2
Optimizing Affinity in HIV-1 Protease Inhibitors
79 BIO429 LESSON 2
En biologie, lutilisation de la thermodynamique est principalement oriente
vers ltude des quilibres et la dfinition de relations entre les proprits
dun systme, mais elle permet galement de dterminer le sens des ractions
dans des systmes qui ne sont pas lquilibre et de caractriser des
processus irrversibles.
La thermodynamique a nanmoins des limites; elle ne nous informe
- ni sur la rapidit dune raction,
- ni sur la manire dont une raction se droule (interactions
- ni sur la manire dont une raction se droule (interactions
molculaires).
Pour obtenir ces informations, il faut avoir recours des tudes cintiques
dans lesquelles on observe lvolution du systme au cours du temps. Les tudes
cintiques fournissent une mesure de la vitesse laquelle une raction
sapproche de son tat dquilibre ou sen carte si la raction est couple une
seconde raction.
Les deux approches sont donc complmentaires et doivent tre exploites
conjointement dans ltude des ractions biologiques
80 BIO429 LESSON 2
The equilbrium constant describes the relative concentrations of components at
equilibrium.
It says nothing about how quickly binding occurs - it could be very slow even if it
is thermodynamically favorable.
However, the ratio of the association and dissociation rates is a function of how
tightly the molecules bind.
At EQUILIBRIUM, the amount of ML does not change with time
81 BIO429 LESSON 2
2.5.7. Surface plasmon resonance (SPR Biacore)
82 BIO429 LESSON 2

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