Transcript Slide 1

Biochemistry 5012: Section on Protein Biochemistry
Lecturer: Chuck Sanders
Lectures 7-8
Protein Stability and Folding
Protein Folding and Disease
Learning Objectives
To understand the basic principles and diversity of protein stability.
To understand the energetic basis for rates of processes: kinetics.
To understand the nature of protein folding.
To survey the relationship of protein folding and human disease.
Topic Outline
Thermodynamic stability of proteins
Stabilizing forces for folded proteins
Kinetic and cellular stability of proteins
Introduction to kinetics
Free energy barriers and rate limiting steps
The nature of protein folding pathways
Mutations and perturbation of protein folding and stability
Protein denaturing agents
Protein stabilizing agents
Protein folding under cellular conditions
Chaperones
Folding Quality control
Protein folding and disease
CFTR and cystic fibrosis
PMP22 and Charcot-Marie-Tooth Disease
Rhodopsin and Retinitis Pigmentosa
Complex disease and non-genetic factors contributing to misassembly
Heritable Genetic Lesions Logged
Protein Folding and Disease
Human Gene Mutation Database (Cardiff)
7000
www.hgmd.org
6000
53,000 known inherited mutations
5000
4000
>50% lead to a single amino acid
change in a protein.
3000
2000
1000
1986
1988
1990
1992
1994
1996
Year
1998
2000
2002
Thermodynamic Stability of Proteins
folded
unfolded
C
Keq,fold
Keq,fold=
[folded]
[unfolded]
o
G

N
Standard Free Energies of Folding for Single Domain Proteins
Protein
Gofold (kcal/mol)
lambda repressor
alpha spectrin SH3 domain
arc repressor
cytochrome C (with heme and Fe(II)
CD2
procarboxypeptidase
U1A spliceosomal protein
Hpr
-3.0
-2.9
-6.3
-15
-8.2
-4.1
-9.3
-4.6
Where Does the Energy Come From to Stabilize Proteins?
How disulfide bonding contributes to protein stability:
folded
unfolded
C
Keq,fold
S
S
S
S
Disulfide bonding
destabilizes
the unfolded state.
N
o
G
no disulfide bond
o
G
with disulfide bond
energy by which disulfide
bond formation destabilizes
the unfolded state by lowering
its conformational entropy
For Globular Water Soluble Proteins:
Interiors are generally hydrophobic.
Interiors dominated by regular 2º structure.
Protein exteriors are polar.
Loops, turns, strands usually exterior.
Balance Sheet for Protein Folding: Ribonuclease T1
Stabilizing:
Destabilizing:
salt bridges: -10 kcal/mol
loss of conformational entropy:
177 kcal/mol (+1.7 kcal per 104 residues)
disulfide bonds: -7 kcal/mol
bury 150 hydrophobic carbons:
(-0.75 kcal/mol each): -112 kcal/mol
replace 104 protein-water hydrogen bonds
with protein-protein and water-water H-bonds
(-1.6 kcal/mol each): -166 kcal mol
Transfer of 73 peptide groups
from water to interior
(+1.1 kcal/mol each): 81 kcal/mol
bury polar groups: 28 kcal/mol
Net: 286 - 295 = -9 kcal/mol in favor of folding
Other Forms of “Protein Stability”
Kinetic Stability (a.k.a. Thermal Stability)
“irreversible”
aggregation
Irreversible means…
Cellular Stability
Proteolyic
Degradation
Described by their
half-lives (time1/2).
Typical t1/2for
cellular stability:
hours to days.
Protein Folding Pathways: Introduction to Kinetics
o
G
2 states
1 barrier in each direction
G*forward G*reverse
G
2 states
2 barriers in each direction
1 intermediate
Some processes have more than one rate barrer, in
which case the barriers are separated by intermidate
states. If a single barrier for one direction of the
reaction is much larger than the others, it is referred
to as the “rate limiting step” of the process.
o
G* : free energy of activation
rate is proportional to rate constant, k,
which is proportional to:
G*
e RT
There is an inverse logrithmic relationship Go
between rate and activation energy.
G*
RT
(the Arrhenius equation)
k=A.e
G*
ln k =
+ ln A (linear form of Ar. eq.)
RT
Intermediate
rate limiting step
to forward reaction
Relationship of Protein Stability to Folding Kinetics
folded
unfolded
C
kfold
kunfold
N
K
eq,fold
o
=
[folded]
[unfolded]
G = -RTlnK eq,fold
=
kfold
kunfold
k are rate constants
“Leventhal’s Paradox
Time required for a 100 residue protein to systematically
explore all possible conformations is:
time = 10100/1013s-1 = 1087 s
The age of the universe is thought to be “only” 1018 s.
Proteins do not fold by a random search of
all possible conformations!
THERE ARE PREFERRED FOLDING PATHWAYS
Protein Folding Pathways
•Secondary structures formed quickly
•Secondary structures form motifs
•Hydrophobic collapse to molten globule
•Final fold of domain is locked in.
•Overall time for folding msec-sec.
Mutations can perturb the heights of the rate
barriers and/or the energies of the free energy
states. Consider a mutation that lowers the
energy of a folding intermediate:
Examples of Potentially
Deadly Mutations
Barrier to folding becomes too
high.
Normal barrier to misfolding is
reduced.
unfolded
G
o
Mutation that lowers
energy of folding
intermediate (only).
folded Folding intermediate is
stabilized.
Folded form is destabilized.
Disrupt interactions with the
protein folding machinery.
o
G
Note that this mutation not only makes the intermediate
much more stable (longer-lived), but that the energy
barriers from this state to both initial and final states
are now larger than for wild type.
Denaturing Proteins
Denaturation = Unfolding
Sodium dodecylsulfate, SDS, is
also a denaturing agent.
Chaotropic Agents:
Unfolding of a protein occurs
when a critical temperature is
exceeded:
Nature of DeNaturation
Denaturation: shift of equilibrium
Denaturation ≠ Aggregation
Denatured protein often does aggregate,
especially at high T.
Protein Stabilization Agents
glycerol
trimethylamine oxide
betaine
Ligand binding also usually stabilizes proteins:
Unfolded protein
Folded Protein + Cognate Ligand
K1
Protein-Ligand Complex
K2
Koverall = K1 x K2
unfolded
Go
folded
folded protein-ligand complex
Protein Folding in the Cell
In vivo
In vitro
lower organisms:
1 gene per protein
diploid organisms:
2 alleles per protein
The main locations of protein folding
in the cell are the cytosol and the
endoplasmic reticulum.
Protein transport in the cell:
(1) Diffusion
(1) Vesicular Trafficking
Protein folding in the cell…
Structure determined by sequence.
Folding pathways differ from test tube:
Folding is often co-translational
Chaperone proteins play roles
Other folding accessory proteins.
Folding is monitored by quality control.
Folding-defective protein is degraded.
Protein Chaperones
•
•
•
•
•
Form complexes with nascent proteins
Prevent aggregation
Favor correct structures
Help proteins across membanes
Chaperones catalyze folding
-Passively
-Actively
Proteins That Assist Protein Folding
Chaperonins
“Chaperones”
Small Heat Shock Proteins
Calnexin/Calreticulin
Petidyl Proline Isomerase
Protein Disulfide Isomerase
Proteases
… And many others
a chaperonin (such as GroEL/ES or hsp60)
Chaperones and Chaperonins
Protein Folding Quality Control Systems
A system for cytoplasmic proteins.
A system for most secreted and membrane proteins.
In The Cytosol: Polyubiquitination and The Proteasome
Protein Folding Quality Control: The Other ER
Relevant Proteins:
Other Facts:
•All secreted proteins
•Cell surface proteins
•Organellar proteins
•ER:>10% of the cellular volume
•25-40% of all proteins
•Quality control in ER
Calnexin Cycle Slides
Unfolded Protein Response (UPR)
Protein Folding, Misfolding
and Disease
Protein Biogenesis is not Always Efficient
Folded
Protein
Ribosome
Nascent
Protein
Misassembled
Protein
Contributions of Protein Misassembly to Human Disease
Simple Disease: Single gene, inherited
Complex Disease: Wrong combination of risk factors.
Sporadic/Acquired Disorders: Non-inherited.
Infectious Disease: Sometimes involves folding-defective protein.
Selected Simple Heritable Disorders
Known to Involve Protein Misassembly
Cystic Fibrosis
CFTR
Charcot-Marie-Tooth
PMP22
Connexin-32
Retinitis Pigmentosa
Rhodopsin
Rom-1
Peripherin
Some Cardiac Arrhythmias
Potassium Channels
Diabetes Insipidus
Aquaporin-2
Vasopressin Receptor
Familial Alzheimer Disease
Beta Amyloid Precursor
Presenilin
The Cystic Fibrosis
Transmembrane
Conductance
Regulator (CFTR)
and
Cystic Fibrosis
The ΔF508 Mutation of CFTR
Peripheral Myelin Protein 22 and Charcot-Marie-Tooth Disease
Myelin Sheath
Surrounding
Axon in Peripheral
Nervous System
Peripheral Myelin Protein 22
In vivo efficiency of folding of wild type PMP22 is ca. 20%.
The efficiency of some disease-related mutants is near 0.
G N V
C S
N
T
H L
W Q 41
H
S S S
W
H
N
C
E
L
G
S
E N P
F
P
D A H N
W 60
122
D
T
G
S
S
H
L
Y
S
R
I V
S
26
Q
V
Y 132
S
Q
W
S
116
T
G
V
I
Y
V Q
F A
I
68
A
A
T
A
V
Y
T
A
S
S
F
M
I L
L
M
L
I L
18
V
V
S
W V A
C
L
I
L A
A
V
A
I
G
F 143
F
V
S
P
L
L
L 11 76
I
H
I
V
Q
F
L L A
S L L
I
I I
S
S
F
100 G
T
G 150
F
L
V
L L L
I
F
I
Y
C
L L
Q
F
V I Y
L
85
H2N- M
L
F
R
G
R K R
T
E -COOH
G
93
160
L T K
Myelin
Membrane
Sites for which point mutations lead to
Charcot-Marie-Tooth Disease
Charcot-Marie-Tooth Disease Involves Defective Myelination in the PNS
Normal
Myelin:
Defective
Myelin
Hypomyelination
Onion Bulbs
Onion Bulbs
Charcot-Marie-Tooth Disease Type 1A (CMTD1A)
Most common cause is third PMP22 allele.
Some forms caused by dominant PMP22 mutations.
PMP22 mutants misfold in the ER and are targeted for ERAD.
Disease symptoms worse for the WT/mutant case than for WT/null.
Explained by PMP22 dimerization in the ER.
There seems to be a “gain of function” component to this disorder…
Peripheral Myelin Protein 22
In vivo efficiency of folding of wild type PMP22 is ca. 20%.
The efficiency of some disease-related mutants is near 0.
G N V
C S
N
T
H L
W Q 41
H
S S S
W
H C
N
E
L
G
S
N
E
P
F
P
D A H N
W
122
D
60
T
G
S
S
H
L
Y
S
R
I V
S
26
Q
V
Y 132
W Q S
S
116
T
G
V
I
Y
V Q
F A
I
68
A
A
T
A
V
Y
T
A
S
S
F
M
I L
L
M
L
I L
18
V
V
S
W V A
C
L
I
L
A
V
A
I
A
G
F 143
F
V
S
P
L
L
L 11 76
I
H
I
V
Q
F
L L A
S L L
I
I I
S
S
F
100 G
T
G 150
F
L
V
L L L
I
F
I Y
Y
L
C
L
Q
F
V I
L
85
H2N- M
L
F
R
G
R K R
T
E -COOH
160
L T K G 93
Myelin
Membrane
Sites for which point mutations lead to
Charcot-Marie-Tooth Disease
Documented
CMTD-1A
Mutations
His12Gln
Leu16Pro
Val30Met
Asp37Val
Ala67Pro
Met69Lys
Ser72Leu
Ser72Pro
Ser72Trp
Ser76Ile
Ser79Cys
Ser79Pro
Leu80Pro
Gly93Arg
Gly100Arg
Gly100Glu
Leu105Arg
Gly107Val
Cys109Arg
Thr118Met
Leu147Arg
Ser149Arg
Gly150Asp
Gly150Cys
Arg157Gly
Arg157Trp
Rhodopsin and Retinitis Pigmentosa
10
H2N- M N G
T>K E
G
P
Y
V
E
G
P
100
F
A
L
I
L
I
L
V
T
T
N
M
V
G>A,R,V
F
P>R
119 L
L
D
79 L
F
L
V
A
L
L
Y 61
V
N
Y
L
Q
P
H
K
K
I
S
A
N
I
G
A
F
130 V
L
L>P
I
V>M
T
T 70
G
V
L>R
H
E
Y
I
T
W
I
Y
V
G
M
S
N
Q
300
250 V
E K
P
F
I
K
V 230
A
P
Y
M
E
F
M
N
310
Q
K
K
C
M
C
C
G
I
T
N
T
K
320
N
L
Q
P
T
V
A
T
A
A
S
A Q Q Q
Sites for which point mutations cause
retinitis pigmentosa or related visual disorders.
A
N
I
K>N ,E,M
R
E
F
A>E
S>R
V
I
T> P
A
Y
R
T
A
F
I
I
V
M
290 I
I
V
M
M
F
T
R
P
L
260 A
F
F
K
C
I
I
270
S>R
V
W
F 221
P
Y
F
I
F> C
C>R
A
P>R,L
211
T
I
E>K
G
150
V
V H>R ,P
M> R,K
I
V
M>R
P> L
161
V
A
F
A
V
Y
V> M
F
Y
F
G
T
F
I
L
N
R>G,L,W
V
M
F
H
N 200
E
S
F
N
Q
N
A> E,V
M G
A
A
C>R
F
A
V
W
S
G
V
Y>N
171
P>Q,L ,S
P> R
A
L
E
I
S
A
C>S 140
R
L
G
280
P
E
R
A
L
F
A
L K
P> A
E>K
G>S
G>D,V
F
T
180
N
E
T
F
Y
S>P
T
C>R
L
G>R 110
Q>P
C>F,Y
L
G
G>D
L
90
V>D
F>L
L>R
50
S
190
Y
D >N,G,Y
G>R,E
C>Y
P
Y
L
T>I
A
Y M> T
L
S
T
S
H
I
G>R,W
Y
39 M L> R
V G T>M A
F
F
Q
N>S
Q>H
Y
A
W
F S
20
30
L
V P
F P>H,A ,L S
R V
E
Y
Y
P N F
E
T A
S A
K
S
240
E
D
L
D G
348
A
330
-COOH
P> A,R,Q,L,S,T
T
340
E
A> P
S
T>M
Q V>L,M
Example of a Rhodopsin Mutations Which Lead to Retinitis Pigmentosa
Pro267Arg
RHOD = rhodopsin
BOPS = blue opsin
VSPR = vasopressin receptor
FSHR = follicle stimulating hormone receptor
TSHR = thyroid stimulating hormone receptor
LHCR = leuteinizing hormone receptor
MC4R = melanocortin 4 receptor
MC2R = melanocortin 2 receptor
ETBR = endothelin B receptor
GRHR = gonadotropin-releasing hormone receptor
retinitis pigmentosa
tritanopia
diabetes insipidus
amenorrhea
various thyroid disorders
sexual development disorders
obesity
glucocorticoid deficiency
Hirschsprung disease
hypogonadotropic hypogonadism
Example of a Rhodopsin Mutation That Leads to Retinitis Pigmentosa:
Pro267Arg
Pro267 site in Rhodopsin
Rhodopsin Mutation That Leads to Retinitis Pigmentosa:
Pro303…
Pro303 site in Rhodopsin
Examples of Complex or Sporadic Disorders
for Which Protein Misassembly can be a Factor
Type II Diabetes
amyloid formation by the islet amyloid peptide
Cancer
mutations in the p53 tumor suppressor protein
Common
Alzheimer’s
Disease
common variants of Apolipoprotein E
Atherosclerosis
mutations in the low density lipoprotein
(LDL) receptor
Protein misassembly may be triggered by gene mutations, but even
wild type protein can misassemble as a result of any of the following
factors:
chemical modification (possibly enzyme-mediated)
heat (as in fever)
cold (as in frostbite)
oxidative stress
toxic agents (including drugs and smoking)
defective protein trafficking
over- or underexpression of protein
kidney dialysis
medical implants
defective protein folding quality control system
inflammation
burns