Transcript lecture 3

3-1
Protein folding in the cell (I)
Basics
- cell compartments, molecular crowding: cytosol, ER, etc.
Folding on the ribosome
- co-translational protein folding
Molecular chaperones
- concepts, introduction
- intramolecular chaperones
- chemical chaperones
- protein chaperones
Cell compartments and folding
3-2
• eukaryotes
- cytosol ..................................protein synthesis, folding/assembly
- extracellular .........................proteins are exported in folded form
- mitochondria ........................limited protein synthesis; energy production
- chloroplasts ..........................limited protein synthesis; light harvesting
- endoplasmic reticulum.......... import of unfolded proteins; protein processing
- peroxisome ........................... import of folded proteins; anab./catab. pathways
- nucleus ................................. import of folded proteins
- lysosome................................import of unfolded proteins; degradation
• bacteria
- cytosol ..................................protein synthesis, etc.
- periplasm .............................import and folding of periplasmic proteins
- extracellular .........................proteins are exported
• archaea
- cytosol ..................................protein synthesis, etc.
- extracellular .........................proteins are exported
3-3
Folding in vitro vs. in vivo
in vitro
in vivo
protein denatured
in a chaotrope
Differences:
folding by dilution
in buffer
folded
protein
1. One has all of the
information immediately
available for folding; the
other process is gradual
2. the cellular
environment is very
different (much more
crowded)
folding
folded
protein
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Co-translational protein folding
Fact:
- first ~30 amino acids of the polypeptide chain
present within the ribosome is constrained
(the N-terminus emerges first)
folding
assembly
Assumption:
as soon as the nascent chain is extruded, it will start
to fold co-translationally (i.e., acquire secondary
structures, super-secondary structures, domains)
until the complete polypeptide is produced and
extruded
3-5
Sindbis Virus Capsid Protein (SCP)
• SCP is the capsid protein of the Sindbis virus
• 26S Sindbis RNA encodes a polyprotein
N
C
SCP E1
E2
E3
• SCP is auto-proteotically cleaved from the rest of the polyprotein
• other cellular proteases cleave E1-E3 from the polyprotein to generate
the mature proteins; E1, the envelope protein, is 9 kDa
• SCP is a 33 kDa serine protease
• WT SCP self-cleaves;
Ser215 => Ala215 mutant doesn’t
catalytic triad &
C-terminus of SCP
3-6
SCP folds co-translationally
Experiment:
1. make and translate different SCP construct RNAs in vitro in the
presence of 35S-methionine for 2 min
2. Prevent re-initiation of translation with aurintricarboxylic acid (ATCA):
‘synchronizing’
3. at set timepoints, add SDS buffer and perform SDS-PAGE
4. observe by autoradiography
2
Result:
*
N
C
SCP E1
C
SCP
C
SCP E1
5
6
7
8
10
12 min
42 kDa
33 kDa
9 kDa
3
4
5
6
7
8
10
12 min
42 kDa
33 kDa
9 kDa
WT
SCP
2
N
4
Mut
SCPE1
2
N
3
WT
SCPE1
3
4
5
6
7
8
10
12 min
42 kDa
33 kDa
9 kDa
Macromolecular crowding
in vitro
E. coli cytosol
~340 mg/ml
<0.1 mg/ml
Ellis and Hartl (1996)
FASEB J. 10:20-26
ribosome
proteins
chaperonin
nucleic acids
other
macromolecules
When doing experiments in vitro, we should all be thinking about this:
proteins in isolated (pure) systems may not behave as they do in the cell
- binding partner(s) might be missing
- post-translational modifications might be missing
- cell conditions (pH, salts, etc.
may be dramatically different
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3-8
Effects of crowding
Definition:
Molecular crowding is a generic term for the condition where a
significant volume of a solution, or cytoplasm for example, is occupied
with things other than water
Fact:
- association constants (ka) increase significantly
- dissociation constants (kd) decrease significantly (kd=1/ka)
- increased on-rates for protein-protein interactions
(see for example Rohwer et al. (2000) J. Biol. Chem. 275, 34909)
Assumption:
- non-native polypeptides will have greater tendency to associate
intermolecularly, enhancing the propensity of aggregation
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Effects of crowding: example
denatured
lysozyme,
reduced or
oxidized
dilution
in buffer
with different
crowding
agents
measure
lysozyme
activity
oxidized
lysozyme
loss of activity
due to protein
aggregation
reduced
lysozyme
crowding agents: ficoll 70*,
dextran 70, protein (BSA, ovalbumin)
*roughly spherical polysaccharide
van den Berg et al. (1999) EMBO J. 18, 6927.
3-10
Problem: non-native proteins
• non-native proteins expose hydrophobic residues that are
normally buried within the ‘core’ of the protein
• these hydrophobic amino acids have a strong tendency to
interact with other hydrophobic (apolar) residues
- especially under crowding conditions
exposed
hydrophobic
residues
X
X
X
intramolecular
incorrect
molecular
interactions
&
loss of activity
X
intermolecular
X
X
X
misfolding
X
X
X
aggregation
3-11
Solution: molecular chaperones
• in the late 1970’s, the term molecular chaperone was coined to
describe the properties of nucleoplasmin:
Nucleoplasmin prevents incorrect interactions between histones and DNA
Laskey, RA, Honda, BM, Mills, AD, and Finch, JT (1978). Nucleosomes are assembled
by an acidic protein which binds histones and transfers them to DNA. Nature 275, 416-420.
Dictionary definition:
1: a person (as a matron) who for propriety accompanies one or more young
unmarried women in public or in mixed company
2: an older person who accompanies young people at a social gathering to
ensure proper behavior; broadly : one delegated to ensure proper behavior
• in the late 1980’s, the term molecular chaperone was used more
broadly by John Ellis to describe the roles of various cellular
proteins in protein folding and assembly
Molecular chaperones:
general concepts
Requirements for a protein to be considered a chaperone:
(1) interacts with and stabilizes non-native forms of protein(s)
- technically also: folded forms that adopt different protein conformations
(2) not part of the final assembly of protein(s)
Functions of a chaperone:
self-assembly refers to the folding of the polypeptide, as well as to
its assembly into functional homo- or hetero-oligomeric structures
“classical”
- assist folding and assembly
assisted
self-assembly
more recent
- modulation of conformation
(as opposed to spontaneous
self-assembly)
- transport
- disaggregation of protein aggregates
- unfolding of proteins
prevention of assembly
assisted
disassembly
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Molecular chaperones:
common functional assays
Type of assay
Rationale
Binary complex
formation
If chaperone has high enough affinity for an unfolded
polypeptide, it will form a complex detectable by:
• co-migration by SEC;
• co-migration by native gel electrophoresis
• co-immunoprecipitation
Prevention of
aggregation
Binding of chaperones to non-native proteins often
reduces or eliminates their tendency to aggregate. Assay
may detect weaker interactions than is possible with SEC
Refolding
Chaperones stabilize non-native proteins; some can assist
the refolding of the proteins to their native state. Usually,
chaperones that assist refolding are ATP-dependent
Assembly
Some chaperones assist protein complex assembly
Activity
Some chaperones modulate the conformation/activity of
proteins
(Miscellaneous)
A number of chaperones have specialized functions
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3-14
Intramolecular chaperones
Concept:
- portions of a polypeptide may assist the biogenesis of the mature
protein without being part of the final folded structure
- these regions are chaperones by definition, although “classical”
molecular chaperones act inter-molecularly, not intra-molecularly.
Intramolecular chaperone: example
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acid-unfolded;
with 77aa propeptide
Subtilisin E
- non-specific protease
- mature protein cannot fold
properly if propeptide is
removed
Gdn-HCl unfolded; with propeptide
Gdn-HCl unfolded;
without 77aa propeptide
propeptide
(77 aa)
precursor (352 aa)
mature protein (275 aa)
Shinde et al. (1993) PNAS 90, 6924.
Intramolecular chaperone: continued
Subtilisin E propeptide
- unstructured alone in solution
- alpha-helical when complexed with
subtilisin? propeptide is ~ 20% of preprotein;
CD suggests combination mature subtilisin
+ propeptide mostly helical
alpha-helical:
minima @ 208, 222 nm
maximum @ 192 nm
- more pronounced
minimum at 208 nm
compared to 222 nm
suggests less helical
Structure
propeptide in TFE
ellipticity
Interpretation
of CD data
propeptide with
subtilisin
alpha
propeptide
beta
coil
subtilisin
nm
beta-sheet:
minimum @ 220 nm
Maximum @ 193 nm
random coil:
maximum ~220 nm
Note:CD traces are additive
 Propeptide must interact with subtilisin
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Intramolecular cleavage or
intermolecular?
Result:
3-17
released
propeptide
Fact: unfolded His10-preprotein
can refold alone in solution
Experiment:
1. prepare subtilisin pre-protein
containing an N-terminal polyhistidine
tag (His10)
2. unfold in denaturant
3. bind different concentrations of the
protein to Ni2+-NTA resin
4. assay for folding by measuring
propeptide release
Q: what do the results mean?
Q: why bind the protein to a resin?
Q: why use different concentrations of
proteins?
full-length
protein
Li et al. (1996) J. Mol. Biol. 262, 591.
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Chemical chaperones
Concept:
- small molecules could enhance the stability and assist the folding
or assembly of proteins
- under conditions of cellular stress, such as a heat-shock, small
molecules may help proteins from misfolding and aggregating
- one easy way to test is to see how they can prevent loss of
activity, or, prevent the aggregation of a protein
- protein aggregation can be conveniently monitored
spectrophotometrically at 360 nm, where light scattering
from the aggregates is detected
3-19
A
Firefly-luc
F-luc in GuHCl
F-luc in GuHCl
B
protein aggregation
in vitro studies
Chemical chaperones: example
Singer and Lindquist (1998) Mol. Cell 1, 639.
Chemical chaperones: example
bacterial luciferase expressed in yeast;
subjected to heat shock conditions
in vivo studies
B
C
40ºC
heat shock
tps1 yeast cells
have a deletion
in the trehalose
synthase
40ºC
heat shock
3-20
without
with
protein aggregation
protein aggregation
Different chemical chaperones
glycerol is often used
to stabilize proteins in vitro
3-21
trans-acting protein molecular
chaperones
- cis-acting (intramolecular) chaperones are relatively rare
- chemical chaperones may play an important role in protecting proteins in
the cell, but their extent of action is likely to be limited
- organisms have evolved large families of protein molecular chaperones
that have either general functions in the cell, or have highly specific
functions
- the expression of many of the chaperones is induced under cellular stress
conditions--giving rise to the name “Heat-shock proteins”, or Hsps,
followed by their Molecular Weight (MW)
BUT:
- not all chaperones are Hsps
- not all Hsps are chaperones
Best characterized: small Hsps (12-42 kDa), Hsp40, Hsp60
(chaperonins), Hsp70, Hsp90, Hsp100/Clp/AAA ATPases
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3-23
Functional proteins from a random-sequence library
Anthony D. Keefe & Jack W. Szostak
Nature 410, 715-718 (2001)
The PDF file of this manuscript is available on the MBB443 web site
There will be one question on the first exam relating to this paper