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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;

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Presentation on theme: "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;"— Presentation transcript:

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 4-1

2 Intramolecular cleavage or intermolecular? Li et al. (1996) J. Mol. Biol. 262, 591. Fact:unfolded His 6 -preprotein can refold alone in solution Experiment: 1. prepare subtilisin pre-protein containing an N-terminal polyhistidine tag (His 6 ) 2. unfold in denaturant 3. bind different concentrations of the protein to Ni-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? from last class 4-2

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) 4-3

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. 4-4

5 Monitoring assembly: SEC A 280 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 Assembly of prefoldin alpha beta Prefoldin hexamer dialyze in buffer large Stoke ’ s radius smaller Stoke ’ s radius 4-5

6 Monitoring assembly: cross-linking BS 3 cross-linker alphabeta Cross-linking example: individual prefoldin subunits Cross-linking agents: Typescomments homo-bifunctionalsame active group hetero-bifunctionaldifferent active group NHS esterreacts with primary amines maleimidesreacts with sulfhydryls (cys) water solublemay be partially memb. soluble membrane permeablemay be totally water insol. cleavableusually with thiols (e.g., DTT) photo-activatednormally hetero-bifunctional => BS 3 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 4-6 - 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. OO R O N O +R’R’NH 2 RN O pH 7-9 H R’R’+N HO NHS ester OO

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!!! Monitoring assembly: scattering methods 4-7

8 Monitoring assembly: BIACORE and ITC 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 k d and n (stoichiometry of binding) 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 k on (association) and k off (dissociation) *Biotin, a small ligand, binds streptavidin protein with very high affinity 4-8

9 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, alpha 2 beta 3, as well as alpha 1 beta 2 - results suggest specific subunit arrangement in hexameric complex, i.e., there is a dimer ‘core’ prefoldin beta alpha 2 beta 3 etc. Fändrich et al. (2000) PNAS 97, 14151. 4-9

10 Molten globules - intermediate conformation assumed by many globular proteins under mildly denaturing conditions 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 characteristics - as determined experimentally using experimental techniques involving hydrogen exchange, small-angle X-ray Scattering (SAXS), circular dichroism, fluorescence spectroscopy, binding of hydrophobic probes, etc. chaperone substrate? 4-10

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

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 4-12

13 Probing protein structure with ANS 1-anilinonaphthalene-8-sulfonic acid - Fluorescence emission maximum and strength changes upon binding to a hydrophobic region(s) of proteins Excitation wavelength (maximum at ~370 nm) Emission wavelength (maximum at ~480 nm) ANS alone ANS + BSA (bovine serum albumin) 4-13

14 Bis-ANS 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 ANS 4-14b

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

16 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. 4-15

17 Background for two papers 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!!! 4-16

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