Protein folding in the cell (II)

1. Protein folding in the cell (II) 4-1 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

2. Intramolecular cleavage or intermolecular? Li et al. (1996) J. Mol. Biol.262, 591. 4-2 from last class 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?

3. Protein assembly 4-3 - 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. Methods of monitoringsubunit association 4-4 • 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.

5. Monitoring assembly: SEC 4-5 beta Assembly of prefoldin dialyze in buffer alpha Prefoldin hexamer A280 large Stoke’s radius smaller Stoke’s radius Elution volume 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

6. Monitoring assembly: cross-linking Cross-linking agents: Types comments • homo-bifunctional same active group • hetero-bifunctional different active group • NHS ester reacts with primary amines • maleimides reacts with sulfhydryls (cys) • water soluble may be partially memb. soluble • membrane permeable may be totally water insol. • cleavable usually with thiols (e.g., DTT) • photo-activated normally hetero-bifunctional => BS3 is bis(sulfosuccinimidyl) suberate, a homo-bifunctional NHS-ester, water-soluble cross-linker => DPDPB is a maleimide cross-linker => SMCC is a hetero-bifunctional NHS ester/ maleimide cross-linker - although 6 amino acids have nitrogen in their side chains, only the epsilon-amine 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. O O H pH 7-9 O HO + R N R’ + N NHS ester O O O O R N R’ NH2 4-6 Cross-linking example: individual prefoldin subunits alpha beta BS3 cross-linker

7. Monitoring assembly:scattering methods 4-7 • 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!!!

8. Monitoring assembly:BIACORE and ITC 4-8 BIACORE Principle of measurement: SPR, or Surface Plasmon Resonance - 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) Isothermal Titration Calorimetry (ITC) Principle of measurement: change in heat - 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

9. Monitoring assembly: Others—IP, mass spectrometry alpha2beta3 prefoldin beta etc. 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) PNAS97, 14151. 4-9 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

10. Molten globules chaperone substrate? 4-10 - 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

11. Lysozyme molten globule 4-11 “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 Similar molten globule states very likely occur in vivo Native state

12. Molten globules inside the cell 4-12 Molten globules might be found: - during co-translational protein folding - before translocation into a membrane - following extrusion through a membrane - following cellular stress

13. Probing protein structure with ANS ANS + BSA (bovine serum albumin) ANS alone 4-13 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

14. Bis-ANS ANS 4-14b 4,4'-dianilino-1,1'-binaphthyl- 5,5'-disulfonic acid - strength of emission is better than ANS - photoincorporation occurs with UV irradiation; - retains fluorescence when covalently bound

15. Defining the potential substrate binding site on GroEL crystal structure of E. coli GroEL (only one of the two stacked rings is shown) bisANS UV x-linking proteolytic digest HPLC separation GroEL e.g., chymotrypsin peptides with bis-ANS derived from apical domain identify fluorescent peptides determine identities mass spectrometry

16. Two competing models forde novo protein folding:stochastic or pathway model? 4-15 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 Cell89, 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.

17. 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!!!