Purification of membrane proteins. University)

Transcription

1 Purification of membrane proteins Dr.Alain Jacquet (Chulalongkorn Dr.Alain Jacquet (Chulalongkorn University)

2 Account for about 40% of the proteins in the cell Receptors, ion channels, transmembrane transporters, signal transducers, ion pumps, free energy transducers, etc. Approx. 60,000 protein structures in the PDB (Protein Data Bank, 2011),about 0.5% integral membrane proteins. Membrane proteins Why? Difficulties: To purify, more problematic than water-soluble proteins: Expressed in low amount Highly hydrophobic, Sticky! (membrane-associated), tendency to form aggregates Often more susceptible to degradation by proteases following solubilization To express recombinant forms To stabilize and to crystallize

3 Proportion of membrane-spanning proteins in the proteome of E. coli and S. cerevisiae associated with different cellular functions. Number of membrane proteins classified according to the number of transmembrane helices Proteins with a cytoplasmic C terminus (Cin) are plotted upwards and those with an extracytoplasmic C terminus (Cout) are plotted downwards.

4 Two main types of membrane proteins Peripheral or extrinsic membrane proteins, which interact with the membrane surface non-covalently by means of electrostatic and hydrogen bonds or with covalent bonds through lipids or GPI anchors. Integral or intrinsic membrane proteins, much more strongly associated with the membrane. Interact with hydrophobic moieties of the phospholipid bilayer. Contain one or more apolar domains that span the lipid bilayer (α-helix but also β-sheet). Type I Integral membrane proteins: C-ter embedded in the cytosol or Type II: N-ter in the cytosol.

5 First purification step: isolate the membrane fraction Disrupt the harvested cells (SEE AFTER) Remove unbroken cells with a low speed centrifugation ( g). To remove cell debris that are not membrane, as well as some cell organelles such as inclusion bodies (If cells are bacteria) or nuclei Collect the membrane fraction by centrifuge the supernatant from the last step with a high speed ultra-centrifugation ( g). This step removes all the soluble proteins. Mammalian cell organelle isolation by differential centrifugation

6 Cell harvesting by scraping. Specific membrane markers are required to follow the fractionation procedure, to confirm the stability of the preparation (ATPase, cadherin, ) All Steps on Ice to prevent proteolysis! Add Protease inhibitors in the buffer Do not use trypsin of course for cell detachment (membrane protein digestions)!!!

7 Cell disruption methods Cell pellet resuspended in a suitable buffer for cell disruption (e.g., PBS). Addition of Dnase to reduce viscosity, useful to add a protease inhibitor cocktail to reduce possible protein degradation.

8 Gentle cell lysis to keep all organelle intact! Cells homogenized by passage through a needle or using tight-fitting glassglass Potter or a Dounce homogenizer (Up to passages of the pestle may be required to achieve sufficient cell breakage)... Dounce homogenizer Potter homogenizer Low speed centrifugation ( g) to prepare a post-nuclear supernatant (PNS). Under gentle conditions of homogenization, 50-60% of a fluid phase marker is recovered in the PNS. The rest, which consists partially of unbroken cells, is lost to the nuclear pellet (NP).

9 Ultracentrifugation to isolate membranes (in the pellet or in some zones using sucrose gradient) Protein density: 1.3 g/cm 3 Membrane density: g/cm 3

10 Peripheral Membrane Protein Extraction Peripheral proteins (non lipid- or GPI-anchored) dissociated using relatively mild techniques to break electrostatic or hydrogen bonds between the peripheral proteins and the membrane, without total membrane disruption. High salts useful to decrease electrostatic interactions between proteins and charged lipids Chaotropic ions disrupt hydrophobic bonds present in the membrane surface and promote the transfer of hydrophobic groups from non-polar environment to the aqueous phase. High ph to disrupting sealed membrane structures without denaturing the lipid bilayer or extracting integral membrane proteins min extraction followed by centrifugation (30 60 min, g) to separate the released peripheral membrane proteins (supernatant) from the remaining lipid bilayer

11 Na 2 CO 3 extracted membrane proteins 2-D gel electrophoresis. Silver staining

12 Summary for membrane isolation

13 Integral Membrane Protein Extraction and Purification Necessity to disrupt the lipid bilayer, which may be achieved with organic solvents, but is more commonly accomplished using detergents.

14 Detergents Detergents: amphipathic substances with a polar (hydrophilic) head group and a nonpolar, (hydrophobic) tail. measurable aqueous solubility as both aggregates and as monomers Classified according to the polar part: nonionic, anionic, cationic, or zwitterionic Some detergents contain both polar and nonpolar faces ; Traditional detergent monomers are generally cone shaped; hydrophilic head groups occupying more molecular space than the linear alkyl chains Lipids generally cylindrical; area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble.

15 Above a certain concentration (critical micellar concentration-cmc) in an aqueous environment: detergent molecules associate to form multimolecular complexes, micelles, with hydrophobic interiors and hydrophilic surfaces. At a concentration = 1-3 x CMC, detergent can solubilize integral membrane proteins CMC inversely related to the size of the alkyl chain, sensitive to both temperature and [salt] Generally, Non ionic detergents disrupt protein-lipid interactions but not protein-protein interactions, contrary to ionic and zwitterionic detergents

16 detergent monomers partitioning into the bilayer. Cooperative detergent-detergent interactions destabilize the bilayer yielding mixed lipid-detergent fragments. Eventually, further detergent addition leads to bilayer dissolution and protein solubilization

17 Which detergent will let membrane proteins soluble, monodisperse, and folded (non denaturing)? Not only one detergent that works for all membrane proteins. Therefore, for each membrane protein isolation: detergents screening and particularly IF little information in the literature on the purification of similar proteins Of course, the choice of detergent(s) will also affect the efficiency of downstream protein purification procedures but also cristallization. Must consider the possibility of detergent exchange

18 Detergent structures

19 Centrifuge at x g at 4 C for 45 min. Solubilization Criteria: Retention of a membrane protein in the supernatant following centrifugation for 60 min at 100,000 x g after extraction.

20 Tendency of a detergent to denature membrane proteins: dependence on the size and charge of the polar headgroup, as well as the length of the alkyl tail (parameters also affecting the CMC and the size of the micelle)

21 Membrane protein extraction kit, detergent screening kit avaible. At least from: GE Healthcare, Qiagen, Pierce For optimal solubilization: Membrane incubation with various [detergent], incubation time, buffer concentration, salt solutions, and temperature conditions But analysis of the pellet too! The structural and functional properties of POI checked during the screening!

22 Removal of Detergents high detergent concentrations often required during the initial extraction of integral membrane proteins affect the stability and subsequent analysis of the isolated membrane proteins but also the purification process excess detergent removed or exchanged for an alternative detergent Choice of technique depends on the unique properties of the detergent used and the concentration range of the protein fraction. Bio-beads SM-2 Polystyrene-divinyl-benzene

23 Efficiency of dialysis CMC and micelle Mw-dependent Detergents with linear alkyl hydrophobic groups (e.g. Triton X-100) have a high micelle Mw non dialyzable whereas detergents with a low micelle Mw and and high CMC (e.g. bile acids and their derivatives) easily removed by dialysis. Detergent solutions can be diluted below their CMC so that micelles disintegrate into monomers which can be dialyzed (use of large excess of detergent-free buffer) Detergents with low CMCs typically removed by adsorption to hydrophobic Beads and detergent bound beads can then be removed by filtration or centrifugation. Gel filtration can be used to separate detergent micelles from protein-detergent complexes and free protein based on size differences..

24

25 Membrane protein purification by chromatographic techniques General considerations Importance of functional assays to detect POI but to be sure about the protein integrity in the presence of detergents during purification No single protocol for obtaining membrane protein purification Membrane proteins usually purified as soluble protein-lipid-detergent complexes in an aqueous environment Possibility to use essentially the same separation techniques as used for watersoluble proteins BUT: -) with detergent present in all solutions (protein-detergent complexes are dynamic and lose detergent molecules in the absence of free detergent). -)Detergent concentrations should be above the CMC but lower than what was used during solubilization (typically in the 0.1% range). -)All the matrices non compatible with the presence of detergents! HIC non compatible AF: some ligands sensitive to detergents (proteins, Abs, ) IEX: avoid using anionic detergents with anion exchange columns, and cationic detergents with cation exchange columns GF: ideal final purification step for membrane proteins to remove aggregates and other impurities according to the size while simultaneously performing buffer exchange.

26 Triton X-100: substantial UV absorbance at 280 nm [protein] often overestimated by UV (use other monitoring). Alternative, CHAPS or CHAPSO for solubilization. Influence on the ph and the charge Detergents with COO- groups (as N-lauryl sarcosinate, bile salts, ) precipitate with divalent cations avoiding this class of detergents in the sample when Ca2+ or Mg2+ (No precipitation with CHAPS and CHAPSO) Carboxylic acid-containing detergents (bile salts and N-lauryl sarcosinate) may not be suitable in protein separation where ph values vary (isoelectric focusing, ph gradient elution from ion-exchange resin, ) Such detergent expected to protonate and become insoluble in aqueous media at weakly acid ph value. Non-ionic and zwitterionic detergents do not move in electrical fields, don't bind to ion exchange resins, and do not contribute to the net charge of macromolecules to which they are bound use of non-ionic or zwitterionic detergents for charge-related separation techniques such as ion-exchange chromatography and preparative electrophoresis

27 A lot of techniques to estimate the quality of purified membrane proteins

28 Nanodisc to solubilize membrane proteins Nanodisc: patch of lipids organized as a bilayer and surrounded by stabilizing proteins (called membrane scaffolding proteins (MSPs)) forming amphipathic helical protein belt. MSP derived from human high-density lipoprotein apoa-1, modified to remove undesired domains and to duplicate other domains to increase the protein s length and, thereby, the perimeter of the ND. MP inside nanodisc

29 Membrane protein isolation in a nanodisc MP transiently solubilized with a detergent in the presence of phospholipids and MSP. After detergent removal, MP simultaneously assembles with phospholipids into a discoidal bilayer with the size controlled by MSP length

30 Soluble membrane protein expression using Nanodisc Proteins expressed in the presence (blue) or absence (red) of Nanodiscs. For the analyzed data set, the overall solubility increased from 17.3 ± 2.2% (in the absence of Nanodisc) to 78.8 ± 3.4% (in the presence of NLPs), notably G proteincoupled receptors (GPCRs) TMS = transmembrane segments.