This document discusses protein folding in the cell. It begins by introducing cellular compartments and the concept of molecular crowding in the cytosol. It then discusses co-translational folding, where proteins begin folding as they are synthesized on the ribosome. Molecular chaperones are introduced as proteins that assist other proteins to properly fold and assemble in the cell. Examples are given of intramolecular and chemical chaperones that can stabilize proteins. The effects of molecular crowding in increasing protein association are also summarized.

1. Protein folding in the cell (I)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
3-1

2. Cell compartments and foldingCell compartments and folding
• 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
3-2

3. FoldingFolding in vitroin vitro vsvs.. in vivoin vivo
folding by dilution
in buffer
protein denatured
in a chaotrope
folded
protein
in vitro in vivo
folding
folded
protein
Differences:
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)
3-3

4. Co-translational protein foldingCo-translational protein folding
folding
assembly
Fact:
- first ~30 amino acids of the polypeptide chain
present within the ribosome is constrained
(the N-terminus emerges first)
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
3-4

5. catalytic triad &
C-terminus of SCP
Sindbis VirSindbis Virusus Capsid Protein (SCP)Capsid Protein (SCP)
• SCP is the capsid protein of the Sindbis virus
• 26S Sindbis RNA encodes a polyprotein
• 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
SCP E1 E2 E3
N C
3-5

6. SCP folds co-translationallySCP folds co-translationally
Experiment:
1. make and translate different SCP construct RNAs in vitro in the
presence of 35
S-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
3Result: 4 5 6 7 8 10 12
Mut
SCP-
E1
WT
SCP
min
33 kDa
2
3 4 5 6 7 8 10 12 min2
WT
SCP-
E1
3 4 5 6 7 8 10 12 min2
9 kDa
42 kDa
33 kDa
9 kDa
42 kDa
33 kDa
9 kDa
42 kDa
3-6
N C
SCP E1
*
N C
SCP E1
N C
SCP

7. in vitro
chaperoninchaperonin
nucleic acidsnucleic acids
E. coli cytosol
~340 mg/ml~340 mg/ml~340 mg/ml~340 mg/ml
proteinsproteins
ribosomeribosome
otherother
macromoleculesmacromolecules
Ellis and Hartl (1996)
FASEB J. 10:20-26
Macromolecular crowdingMacromolecular crowding
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 - cell conditions (pH, salts, etc.
- post-translational modifications might be missing may be dramatically different
<0.1 mg/ml<0.1 mg/ml<0.1 mg/ml<0.1 mg/ml
3-7

8. Effects of crowdingEffects 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
3-8

9. oxidized
lysozyme
reduced
lysozyme
loss of activity
due to protein
aggregation
Effects of crowding:Effects of crowding: exampleexample
van den Berg et al. (1999) EMBO J. 18, 6927.
dilution
in buffer
with different
crowding
agents
measure
lysozyme
activity
measure
lysozyme
activity
denatured
lysozyme,
reduced or
oxidized
crowding agents: ficoll 70*,
dextran 70, protein (BSA, ovalbumin)
*roughly spherical polysaccharide
3-9

10. Problem:Problem: non-native proteinsnon-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
intramolecular
misfolding
X
X
X
X
intermolecular
aggregation
X
X
X
X
X
X
incorrect
molecular
interactions
&
loss of activity
exposed
hydrophobic
residues
3-10

11. Solution:Solution: molecular chaperonesmolecular 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
3-11

12. Molecular chaperones:Molecular chaperones:
general conceptsgeneral 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:
“classical”
- assist folding and assembly
more recent
- modulation of conformation
- transport
- disaggregation of protein aggregates
- unfolding of proteins
assisted
self-assembly
(as opposed to spontaneous
self-assembly)
assisted
disassembly
prevention of assembly
self-assembly refers to the folding of the polypeptide, as well as to
its assembly into functional homo- or hetero-oligomeric structures
3-12

13. Molecular chaperones:Molecular chaperones:
common functional assayscommon 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
3-13

14. Intramolecular chaperonesIntramolecular 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.
3-14

15. Intramolecular chaperone:Intramolecular chaperone: exampleexample
Subtilisin E
- non-specific protease
- mature protein cannot fold
properly if propeptide is
removed
Shinde et al. (1993) PNAS 90, 6924.
precursor (352 aa)
propeptide
(77 aa)
mature protein (275 aa)
3-15
Gdn-HCl unfolded;
without 77aa propeptide
Gdn-HCl unfolded; with propeptide
acid-unfolded;
with 77aa propeptide

16. Intramolecular chaperone:Intramolecular chaperone: continuedcontinued
nm
ellipticity
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
propeptide
propeptide with
subtilisin
subtilisin
propeptide in TFE
Note:CD traces are additive
alpha
beta
coil
Interpretation
of CD data
alpha-helical:
minima @ 208, 222 nm
maximum @ 192 nm
- more pronounced
minimum at 208 nm
compared to 222 nm
suggests less helical
Structure
beta-sheet:
minimum @ 220 nm
Maximum @ 193 nm
random coil:
maximum ~220 nm
3-16
 Propeptide must interact with subtilisin

17. Intramolecular cleavage orIntramolecular cleavage or
intermolecular?intermolecular?
Li et al. (1996) J. Mol. Biol. 262, 591.
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
Result:
Q: what do the results mean?
Q: why bind the protein to a resin?
Q: why use different concentrations of
proteins?
3-17
full-length
protein
released
propeptide

18. Chemical chaperonesChemical 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
3-18

19. Chemical chaperones:Chemical chaperones: exampleexample
Singer and Lindquist (1998) Mol. Cell 1, 639.
A
B
proteinaggregation
3-19
invitrostudies
F-luc in GuHCl
F-luc in GuHCl
Firefly-luc

20. invivostudies
B
C
Chemical chaperones:Chemical chaperones: exampleexample
tps1 yeast cells
have a deletion
in the trehalose
synthase
40ºC
heat shock
40ºC
heat shock
3-20
bacterial luciferase expressed in yeast;
subjected to heat shock conditions

21. Different chemical chaperonesDifferent chemical chaperones
without
with
proteinaggregationproteinaggregation
glycerol is often used
to stabilize proteins in vitro
3-21

22. transtrans-acting protein molecular-acting protein molecular
chaperoneschaperones
- 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
3-22

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
3-23