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

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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

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Folding Folding in vitroin vitro vs vs.. in vivoin vivo

folding by dilutionin buffer

protein denaturedin a chaotrope

foldedprotein

in vitro in vivo

folding

foldedprotein

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)

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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

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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 E3N C

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SCP folds co-translationallySCP folds co-translationallyExperiment:1. make and translate different SCP construct RNAs in vitro in the presence of 35S-methionine for 2 min2. Prevent re-initiation of translation with aurintricarboxylic acid (ATCA): ‘synchronizing’3. at set timepoints, add SDS buffer and perform SDS-PAGE4. observe by autoradiography

3Result: 4 5 6 7 8 10 12Mut

SCP-E1

WTSCP

min

33 kDa

2

3 4 5 6 7 8 10 12 min2

WTSCP-

E1

3 4 5 6 7 8 10 12 min2

9 kDa

42 kDa

33 kDa9 kDa

42 kDa

33 kDa9 kDa

42 kDa

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N CSCP E1

*

N CSCP E1

N CSCP

in vitro

chaperoninchaperoninnucleic acidsnucleic acids

E. coli cytosol

~340 mg/ml~340 mg/ml~340 mg/ml~340 mg/ml

proteinsproteinsribosomeribosome

otherothermacromoleculesmacromolecules

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

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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

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oxidizedlysozyme

reducedlysozyme

loss of activitydue to proteinaggregation

Effects of crowding: Effects of crowding: exampleexample

van den Berg et al. (1999) EMBO J. 18, 6927.

dilutionin bufferwith differentcrowdingagents

measure lysozyme activity

measure lysozyme activity

denaturedlysozyme,reduced oroxidized

crowding agents: ficoll 70*,dextran 70, protein (BSA, ovalbumin)

*roughly spherical polysaccharide

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Problem:Problem: non-native proteins 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

intramolecular

misfolding

XX

X

X

intermolecular

aggregation

X

X

X

X

X

X

incorrectmolecular

interactions&

loss of activity

incorrectmolecular

interactions&

loss of activity

exposedhydrophobic

residues

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Solution:Solution: molecular chaperones 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 company2: 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

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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

assistedself-assembly(as opposed to spontaneousself-assembly)

assisteddisassembly

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

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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

RefoldingChaperones 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

ActivitySome chaperones modulate the conformation/activity of proteins

(Miscellaneous) A number of chaperones have specialized functions

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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.

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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)

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Gdn-HCl unfolded;without 77aa propeptide

Gdn-HCl unfolded; with propeptide

acid-unfolded;with 77aa propeptide

Intramolecular chaperone: Intramolecular chaperone: continuedcontinued

nm

elli

ptic

ity

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

betacoil

Interpretationof CD dataalpha-helical:minima @ 208, 222 nmmaximum @ 192 nm- more pronouncedminimum at 208 nmcompared to 222 nmsuggests less helicalStructure

beta-sheet:minimum @ 220 nmMaximum @ 193 nm

random coil:maximum ~220 nm

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Propeptide must interact with subtilisin

Intramolecular cleavage or Intramolecular cleavage or intermolecular?intermolecular?

Li et al. (1996) J. Mol. Biol. 262, 591.

Fact: unfolded His10-preproteincan refold alone in solution

Experiment:1. prepare subtilisin pre-protein containing an N-terminal polyhistidine tag (His10)2. unfold in denaturant3. bind different concentrations of the protein to Ni2+-NTA resin4. 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?

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full-lengthprotein

releasedpropeptide

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

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Chemical chaperones: Chemical chaperones: exampleexample

Singer and Lindquist (1998) Mol. Cell 1, 639.

A

B

pro

tein

ag

gre

gat

ion

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in v

itro

stud

ies

F-luc in GuHCl

F-luc in GuHCl

Firefly-luc

in v

ivo

stud

iesB

C

Chemical chaperones: Chemical chaperones: exampleexample

tps1 yeast cells have a deletionin the trehalose synthase

40ºCheat shock

40ºCheat shock

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bacterial luciferase expressed in yeast;subjected to heat shock conditions

Different chemical chaperonesDifferent chemical chaperones

without

with

pro

tein

ag

gre

gat

ion

pro

tein

ag

gre

gat

ion

glycerol is often usedto stabilize proteins in vitro

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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

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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

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