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4-1
Protein folding in the cell (II)
Protein assembly
- significance; methods used to measure
The molten globule
- what it is, how we measure it, why its important; ANS and bis-ANS
Protein folding in the cell: two competing models
- important literature
Intramolecular cleavage or
intermolecular?
Result:
Fact: unfolded His6-preprotein
can refold alone in solution
Experiment:
1. prepare subtilisin pre-protein
containing an N-terminal polyhistidine
tag (His6)
2. unfold in denaturant
3. bind different concentrations of the
protein to Ni-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?
Li et al. (1996) J. Mol. Biol. 262, 591.
4-2
4-3
Protein assembly
- greater than 50% of all cellular proteins assemble into higher-order structures
- either oligomers of identical proteins (homo-oligomers) or of different proteins
(hetero-oligomers)
- activity of most oligomers strictly depend on their proper assembly
- example of what can go wrong:
- von Hippel-Lindau protein (VHL) assembles with Elongin B and Elongin C in
the cell. Mutations that abrogate assembly lead to cancer
Why protein assembly? why not make larger proteins?
There are some proteins that are really huge, but these are typically more rare (e.g.,
titin, 3 Mda, a skeletal protein). The average size of a protein in E. coli is ~40 kDa
Possible reasons:
- larger protein means more chances of incorporating mutation(s) in the gene, or
having translation error(s)
- symmetric arrangement of subunits preferred over non-symmetric arrangement
(i.e. oligomer versus single protein)
- folding of large proteins may be problematic (i.e., ATP-binding proteins)
4-4
Methods of monitoring
subunit association
• Size Exclusion Chromatography (SEC)
• Cross-linking
• Scattering methods
• BIACORE
• Isothermal Titration Calorimetry (ITC)
• Fluorescence Resonance Energy Transfer (FRET), also called RET
• Other: activity, immunoprecipitation, mass spectrometry, etc.
4-5
Monitoring assembly: SEC
beta
Assembly of prefoldin
dialyze in buffer
alpha
Prefoldin
hexamer
A280
large
Stoke’s
radius
Elution volume
smaller
Stoke’s
radius
Note: Elution volume of a protein / protein complex depends on Stoke’s radius
- analytical ultracentrifugation (equilibrium sedimentation) yields molecular weights
that are irrespective of the shape of the molecule
Monitoring assembly: cross-linking
4-6
Cross-linking agents:
Cross-linking example:
individual prefoldin subunits
alpha
BS3
cross-linker
Types
• homo-bifunctional
• hetero-bifunctional
• NHS ester
• maleimides
• water soluble
• membrane permeable
• cleavable
• photo-activated
beta
comments
same active group
different active group
reacts with primary amines
reacts with sulfhydryls (cys)
may be partially memb. soluble
may be totally water insol.
usually with thiols (e.g., DTT)
normally hetero-bifunctional
=> BS3 is bis(sulfosuccinimidyl) suberate, a homobifunctional NHS-ester, water-soluble cross-linker
=> DPDPB is a maleimide cross-linker
=> SMCC is a hetero-bifunctional NHS ester/ maleimide
cross-linker
O
pH 7-9
O
R
O
N
NHS ester
O
+ R’
NH2
O
R
O
H
N
R’+ HO
N
O
- although 6 amino acids
have nitrogen in their side
chains, only the epsilonamine of lysine
cross-reacts significantly
with NHS esters. Note that
Tris (often used as a buffer)
also has a primary
amine, and cross-reacts
with NHS esters.
Monitoring assembly:
scattering methods
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• Light scattering: 320 nm for measuring non-specific ‘assembly’, i.e., protein aggregation
• dynamic light scattering:
- same principle, except use specialized equipment (laser, detector)
- can obtain information regarding the stoke’s radius of a protein / protein complex
- if protein is globular, can estimate molecular weights
- can obtain information on the distribution of particle sizes
- uses relatively high protein conc’n of > 0.1 mg/ml (potentially problematic)
- experiments performed very quickly
• Small-Angle X-ray Scattering (SAXS):
- measures radius of proteins / protein complexes
- new advances allow for the detection of some domain structure
(e.g., can see ‘hole’ in GroEL double-ring structure by applying
sophisticated algorithms to the SAXS data); need lots of protein though!!!
Monitoring assembly:
BIACORE and ITC
4-8
BIACORE
Isothermal Titration Calorimetry (ITC)
Principle of measurement:
SPR, or Surface Plasmon Resonance
Principle of measurement:
change in heat
- one of the proteins must be physically attached to
the BIACORE chip surface, either covalently by
cross-linking or with affinity tag (e.g., polyhis,
biotin* label); the other protein is free in solution.
Note: best to do the reverse experiment also!
- instrument measures refractive index change
- measure kon (association) and koff (dissociation)
- one concentrated sample (protein, ligand, etc.)
is injected into chamber containing the second
reactant (e.g., protein)
- a titration with increasing sample is performed
- heat which is evolved is detected by instrument
- measure kd and n (stoichiometry of binding)
*Biotin, a small ligand, binds streptavidin
protein with very high affinity
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Monitoring assembly:
Others—IP, mass spectrometry
Immunoprecipitation
- lyse cells
- add primary antibody which binds protein of interest
- add secondary antibody coupled to beads
- detect protein(s) bound precipitated protein, using
radiolabeled cells or by Western blot detection
Mass spectrometry
- normally, can detect masses of individual proteins
in a complex
- using nanoflow electrospray coupled to TOF MS,
it is sometimes possible to detect protein
complexes by using less energy
- in example, see prefoldin hexamer at low
energies, and various dissociated subunits at
higher energies: beta alone, alpha dimer,
alpha2beta3, as well as alpha1beta2
- results suggest specific subunit arrangement in
hexameric complex, i.e., there is a dimer ‘core’
Fändrich et al. (2000) PNAS 97, 14151.
4-10
Molten globules
- intermediate conformation assumed by many globular
proteins under mildly denaturing conditions
- as determined experimentally using experimental techniques
involving hydrogen exchange, small-angle X-ray Scattering
(SAXS), circular dichroism, fluorescence spectroscopy, binding of
hydrophobic probes, etc.
characteristics
• presence of substantial content of secondary structure
• absence of most of the specific tertiary structure associated with
tight packing of side-chains
• dynamic features of the structure with motions on a timescale
longer than nanoseconds
• the protein is still compact, but radius is 10-30% larger compared
with that of native state
• presence of loosely-packed hydrophobic core
• greater exposure of apolar side-chains
chaperone
substrate?
4-11
Lysozyme molten globule
“molten globule states”
notice how two different
folding paths converge
into a local minimum
(lower energy state)
that is close to, but has
not reached, the lowest
energy (folded) state
Native state
Similar molten
globule states
very likely occur
in vivo
4-12
Molten globules inside the cell
Molten globules might be found:
- during co-translational protein folding
- before translocation into a membrane
- following extrusion through a membrane
- following cellular stress
Probing protein structure
with ANS
1-anilinonaphthalene-8-sulfonic acid
Excitation wavelength
(maximum at ~370 nm)
Emission wavelength
(maximum at ~480 nm)
- Fluorescence emission maximum and strength changes upon
binding to a hydrophobic region(s) of proteins
4-13
4-14b
Bis-ANS
4,4'-dianilino-1,1'-binaphthyl- 5,5'-disulfonic acid
ANS
- strength of emission is better than ANS
- photoincorporation occurs with UV irradiation;
- retains fluorescence when covalently bound
Defining the potential substrate
binding site on GroEL
crystal structure
of E. coli GroEL
(only one of the
two stacked rings
is shown)
GroEL
bisANS
UV x-linking
proteolytic digest
HPLC separation
e.g., chymotrypsin
identify fluorescent
peptides
determine
identities
mass
spectrometry
peptides with bis-ANS
derived from apical domain
4-15
Two competing models for
de novo protein folding:
stochastic or pathway model?
2 papers will be examined:
Farr et al. (1997)
Chaperonin-mediated folding in the eukaryotic cytosol proceeds through
Rounds of release of native and nonnative forms
Cell 89, 927-937.
Siegers et al. (1999)
Compartmentation of protein folding in vivo: sequestration of non-native
polypeptide by the chaperonin-GimC system
EMBO J. 18, 75-84.
Background for two papers
4-16
Chaperonins. Large, double-ring structures that accept non-native proteins inside their
cavities and assist protein folding. The eukaryotic cytosolic chaperonin is involved in
folding actins and tubulins.
Prefoldin. Hexameric molecular chaperone also involved in actin and tubulin
biogenesis. Its existence was not known when the Cell paper was published in 1997 (it
was discovered in 1998). It is also known as Gim complex, or GimC.
Stochastic model for de novo protein folding. The definition of stochastic is: involving
or containing random variables. In this context, it means that folding polypeptides will
interact with whichever molecular chaperone may be present at any one time during its
synthesis. If the protein has not folded or assembled after interaction with chaperone(s),
it will be released in the bulk cytosol and may interact with other chaperone(s) before it
has a chance to fold/assemble.
Pathway model for de novo protein folding. Hypothesis that many newly-synthesized
cellular proteins follow an ordered pathway during de novo protein folding. This
ordered pathway would typically occur when one or more molecular chaperones
interact with the newly made protein.
BUT NOTE THAT MOST PROTEINS can probably fold in vivo either with
minimal or no assistance from molecular chaperones!!!