WO2006036772A2 - Cristallisation de proteines membranaires dans des gels bicouches lipidiques assembles en trois dimensions - Google Patents

Cristallisation de proteines membranaires dans des gels bicouches lipidiques assembles en trois dimensions Download PDF

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WO2006036772A2
WO2006036772A2 PCT/US2005/034098 US2005034098W WO2006036772A2 WO 2006036772 A2 WO2006036772 A2 WO 2006036772A2 US 2005034098 W US2005034098 W US 2005034098W WO 2006036772 A2 WO2006036772 A2 WO 2006036772A2
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protein
crystallization
gel
clb
lipid
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WO2006036772A3 (fr
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Robert M. Glaeser
Shahab Rouhani-Manshadi
Christopher Lunde
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to three-dimensionally connected lipid bilayer (CLB) gel compositions and methods of preparation and use thereof for the crystallization of membrane proteins.
  • CLB lipid bilayer
  • Membrane proteins are estimated to comprise about 1/3 of most cellular genomes.
  • Membrane receptors, small- molecule (and drug) trans-membrane pumps and transporters, and ion channels represent arguably the most sought-after targets for drug development. Advances in biochemistry, cell biology, and drug development will thus be greatly accelerated, once methods are developed that lead to crystallization and high-resolution structure determination at a rate comparable to what is now possible for soluble proteins.
  • the idea that hydrated lipid-bilayer gels are a suitable medium for crystallization of membrane proteins was first introduced by Landau, E. M. & Hosenbusch, J. P.
  • Phosphatidylethanolamine (PE) and certain derivatives of PE represent a class of lipids that forms CLB gels, again consisting of three-dimensionally connected bilayers (Sjolund, M., Lindblom, G., Rilfors, L. & Arvidson, G., Hydrophobic molecules in lecithin- water systems.
  • CLB gels again consisting of three-dimensionally connected bilayers
  • PE-based lipid bilayers are an attractive alternative to monoglyceride bilayers since the diacyl chain and the phospholipid head group must surely present a more natural environment for most membrane proteins.
  • PE-based lipids require rather extreme thermal cycling to reach a stable, three-dimensionally connected bilayer-gel phase (Shyamsunder, E., Gruner, S. M., Tate, M. W., Turner, D. C, So, P. T. & Tilcock, C. P. (1988) Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes., Biochemistry. 27, 2332-6; Tenchov, B., Koynova, R. & Rapp, G. (1998) Accelerated formation o:f cubic phases in phosphatidylethanolamine dispersions, Biophys J. 75, 853-66).
  • PE CLB gels are unstable wh.en equilibrated with excess water. Unlike monoglyceride gels, PE gels swell without limit and thus both the optical clarity and the 3-D connectivity of the gel system is lost. The problem that must be addressed, therefore, is how to equilibrate the gel with buffer solutions that are meant to induce protein crystallization without disrupting the gel itself.
  • compositions and methods of using a fusogen such as diacylglycerophospholipids conjugated to methoxy polyethylene glycol, to achieve facile formation of a stable hydrated CLB gel for the crystallization of proteins.
  • a fusogen such as diacylglycerophospholipids conjugated to methoxy polyethylene glycol
  • compositions and methods of equilibrating gels which result in stable gels to induce protein crystallization.
  • the invention provides for three dimensionally connected lipid bilayer gels for protein crystallization comprising a crystallization mixture of 30-99% aqueoms solution and 1- 70% lipid phase, wherein the lipid phase comprising of about 50-99.9% host lipid and about 0.1 - 50% fusogen, wherein said crystallization mixture is combined with a protein to be crystallized.
  • the host lipid is selected from the group consisting of diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof.
  • the host lipid is a diacylglycerophospholipid selected from the group consisting of saturated or unsaturated phosphatidylethanolamines, mono-methyl phosphatidylethanolamines and derivatives thereof whose fatty-acid chain length is between 14 to 22 carbons long.
  • the host lipid is a diacylglycerophospholipid is selected from the group consisting of di-[18:0]-PE, di-[18:l]-PE, di-[18:2]-PE, di-[16:0]- PE, di-[16:l]-PE, di-[16:0]-N-mono-methyl PE, di-[18:l]-N-mono-methyl PE.
  • the host lipid is a monoacylglycerophospholipid such as lyso-PE.
  • the fusogen is selected from the group consisting of functionalized lipids, monoglycerides, fusogenic lipids, detergents and high protein concentrations of membrane proteins, which promote the formation of the CLB under conditions that retain or confer protein stability.
  • the fusogen is mono- olein or phosphatidylethanolamine conjugated to polyethylene glycol.
  • the fusogen is l ⁇ -dioleoyl-sn-glycerol-S-phosphatidylethanolamine- ⁇ - methoxy polyethylene glycol (DOPE-mPEG).
  • the crystallization mixture further comprises lipid phase additives.
  • the lipid phase additive can be selected from the group consisting of detergents, membrane lipids and membrane components.
  • the crystallization mixture further comprises a crystallization solution.
  • the crystallization solution can be comprised of salts, osmolytes and precipitants, which are specifically formulated based on the structural stability and/or likely lateral association character of the membrane protein of interest.
  • the invention further provides for a method of crystallizing membrane or membrane- associated proteins comprising: (1) providing a three dimensionally connected lipid bilayer gel for protein crystallization comprising a crystallization mixture of 30-99% aqueous solution and 1- 70% lipid phase, wherein the lipid phase comprising of about 50-99.9% dry host lipid and about 0.1 - 50% fusogen; (2) hydrating the crystallization mixture with an appropriate volume ratio of protein, wherein the protein is in aqueous solution; (3) forming the CLB gel; (4) dispensing the gel in a container to promote or allow crystallization and for detection or visualization of protein crystals; (5) overlaying the CLB gel with a crystallization solution to promote or allow crystallization; (6) allowing the CLB gel to sit for various extended periods of time for protein crystals to form.
  • the formation of the CLB gel in step (3) can be performed in a container.
  • the method can further comprise the step of flattening the CLB gel aliquot to improve optical quality and prevent overhydration.
  • the gel aliquot can be flattened by centrifugation or by a coverslip.
  • the invention also provides a process for forming a three-dimensional connected lipid bilayer gel suitable for stability and lateral association of proteins comprising the steps of: (1) preparing a crystallization mixture of 30-99% aqueous solution and 1- 70% lipid phase, wherein the lipid phase comprising of about 50-99.9% host lipid and about 0.1 - 50% fusogen, wherein said host lipid is selected from the group consisting of diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof; (2) combining the crystallization mixture with an appropriate volume ratio of protein; (3) forming the CLB gel and allowing protein to crystallize.
  • the process can further comprise the steps of: (4) dispensing said gel in a container to promote or allow crystallization and for detection or visualization of protein crystals; (5) overlaying the CLB gel with a crystallization solution to promote or allow crystallization; and (6) allowing the CLB gel to sit for various extended periods of time for protein crystals to form.
  • the process wherein the crystallization mixture further comprises a lipid phase additive is selected from the group consisting of detergents, membrane lipids and membrane components.
  • the crystallization mixture can further comprise a crystallization solution comprising salts, osmolytes and precipitants.
  • the process further comprises the step of performing a stability assay.
  • the stability assay is an intrinsic tryptophan fluorescence assay on the CLB gel, wherein the assay is performed by (a) excitation of the CLB gel at about 280 nm wavelength and observing the fluorescence emission spectra from 300-400 nm wavelength; (b) heating the gel to protein denaturing temperatures; (c) excitation of the CLB gel at about 280 nm wavelength and observing the fluorescence emission spectra from 300- 400 nm wavelength for any changes in the spectra from step (a), wherein the changes in the spectra indicate protein denaturation.
  • the invention also provides for a method for promoting CLB stability and the formation of protein crystals, comprising the following steps: (a) combining a crystallization solution with the crystallization mixture of claim 1, (b) combining an appropriate volume ratio of protein with the crystallization mixture of claim 1; and (c) forming the CLB gel.
  • Step (b) can be performed before step (a).
  • the method can further comprise the steps of: (d) dispensing the gel in a container to promote or allow crystallization and for detection or visualization of protein crystals; and (e) allowing the CLB gel to sit for various extended periods of time for protein crystals to form.
  • FIGURE 1 is a cartoon that illustrates how the structure of the pn3m cubic phase provides a three-dimensionally connected lipid bilayer system when the hydrophobic rim of one face of the "tetrahedral unit" is docked to a second such tetrahedral unit.
  • FIGURE 2 is a photograph of the apparatus used to reconstitute membrane proteins into lipid-bilayer gels.
  • A Anhydrous lipid is loaded into one syringe and aqueous membrane protein is loaded into the other syringe. The two syringes are then coupled with the connector described by Cheng et al. (Cheng, Hummel et al. 1998).
  • B The contents of the two syringes are then passed repeatedly through the connector until homogeneous mixing is complete. A thumb operated ratchet is then used to dispense small aliquots of gel into microplate wells.
  • FIGURE 3 is a graph showing the absorption spectrum of bacteriorhodopsin in detergent (octylglucoside) and in mono-olein (MO). Both have a peak at 550 nm, but the spectrum in MO is more broad and there is a small peak at -410 nm implying the presence of a species with a deprotonated Schiff base.
  • the protein in detergent does have a small peak at ⁇ 380 nm due to free retinal that is released from denatured protein. Essentially all of the protein is denatured (retinal is released) when the protein is heated.
  • the spectral curves have been adjusted for background and scaled in order to facilitate comparisons of their respective waveforms.
  • the spectra are identified as: purple membranes (- ⁇ -), detergent-solubilized bR (-T-), bR in MO (— ), heat-treated bR in MO (--).
  • FIGURE 4 is a photograph of the small angle x-ray diffraction pattern of a hydrated gel of monomethyl-PE produced with the syringe apparatus shown in Figure 2.
  • FIGURE 5 is a set of graphs showing both absorption spectra in the visible and fluorescence spectra in the near UV.
  • Absorption spectra of wt bR and a triple mutant of bR demonstrate that wt bR is in a more native conformation in a PE- based gel (A) and that even the very labile "triple mutant" of bR is highly protected against denaturation by the PE-based gel (B).
  • the intrinsic tryptophan fluorescence (280 nm excitation) of bR triple mutant is compared between protein in MO (C) and PE-based gel (D). The spectra are identified as: before heating ( — ) and after heating ( — ).
  • FIGURE 6 is a photograph showing the first crystals of wild-type bacteriorhodopsin, grown in a PE-based lipid gel. The "rectangular" crystals are believed to be hexagonal crystals, viewed on edge.
  • An ideal lipid-bilayer gel for crystallization of membrane proteins is one for which: (1) the native structure of the membrane protein should be stable when reconstituted into the bilayer gel; (2) reconstitution of the protein into the bilayer gel should be facile under protein-friendly conditions; (3) the gel should retain good optical quality when overlaid with or when made with a wide range of crystallization solutions.
  • the connected lipid bilayer (CLB) gels of the invention are formed by lipid bilayers that are curved everywhere as a saddle ( Figure 1). Since such bilayers form a three- dimensionally connected lattice, a protein such as bacteriorhodopsin, can be inserted into the bilayer where it diffuses throughout the volume of the gel. In the presence of appropriate buffer conditions, the protein molecules collide, nucleate, and grow into 3-D crystals. Mono- olein has been a favored lipid to use because formation of the CLB is extremely facile. However, hydrated mono-olein gels have not been productive for the crystallization of other types of membrane proteins.
  • mutants of bacteriorhodopsin are shown, in the examples infra, to be less stable when reconstituted into a mono-olein membrane than they are in the detergent-solubilized state, which is the opposite of what was expected.
  • the CLB gels are comprised of a crystallization mixture of about 30-99% aqueous solution and between 1-70% lipid phase.
  • the lipid phase is comprised of between 50 — 99.9% host lipid, and about 0.1 — 50% fusogen, which can then be mixed with the purified protein to be crystallized. It is preferred that the mixing is performed using the syringe-based mixing technique as described by Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. (1998) A simple mechanical mixer for small viscous lipid-containing samples, Chem & Phys Lipids. 95, 11-21, which is hereby incorporated by reference.
  • the CLB may further comprise lipid phase additives or crystallization solutions which further allow the protein to crystallize and stabilize in the CLB gels.
  • the crystallization mixture to form the CLB gels of the invention is substantially comprised of from about 50 - 99.9% host lipid, which is hydrated with aqueous solution.
  • the host lipid should not only form the CLB gels of the invention but also sufficiently stabilize the protein to be crystallized.
  • Lipids that can be used as the gel- forming host lipids include, but are not limited to, diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof.
  • the types of diacylglycerophospholipids used in the invention include but are not limited to phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, phosphatidylinositols, and phosphatidylglyercols, and derivatives thereof with saturated or unsaturated fatty-acid chains.
  • Chain lengths can vary from 14 to 22 carbons, more preferably 16-18 carbons long. This carbon chain length is generally preferred because the lipid tails in biological membranes are generally this length and one factor in choosing the appropriate host lipid is how closely the lipid mimics a protein's natural environment in the crystallization setup.
  • the host lipids described herein may form the CLB at varying temperatures, dependent upon the length of the lipid tail, the degree of saturation of the lipid tail and the nature of the lipid head group. For example, some host lipids 16 carbons long will form the CLB at room temperatures, while a host lipid 18 carbons long may only form the CLB at room temperature if the lipid head group is modified.
  • the preferred host lipid should have at least one unsaturated bond to permit the formation of the CLB at room temperature.
  • the nature or methylation of the lipid head group may affect the temperature at which the lipid phase is formed. It may be preferred in some embodiments that a lipid head group be unmethylated, however, should the carbon chain of the lipid tail be lengthened, methylation of the head group or lower temperatures may be required to allow the host lipid to form the CLB.
  • the host lipid is selected from the group consisting of di-[18:0, 18:1, 18:2, 16:0, and 16:1]-PE, di-[16:0 and 18:l]-N-mono-methyl PE, and lyso-PE, as the host lipid.
  • host lipids are di-18:l PE and di-18:l- N-mono-methyl PE because they form a stable CLB at 4 0 C and could prove useful for proteins that are not stable at room temperature for extended periods of time.
  • Fusogens A major drawback of diacyl-lipids has been that it is kinetically difficult for them to reach the CLB.
  • the fusogen is a functionalized lipid, monoglyceride, polyethylene glycols, polyethylene glycol-conjugated compounds.
  • functionalized lipids include phosphatidylethanolamines conjugated to methoxy polyethylene glycol (mPEG), such as l ⁇ -dioleoyl-sn-glycerol-S-phosphatidylethanolamine conjugated to polyethylene glycol (DOPE-PEG) , 1 ⁇ -dioleoyl-sn-glycerol-S-phosphatidylethanolamine-N- methoxy polyethylene glycol 550 (DOPE-mPEG550) and and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-methoxy polyethylene glycol 550 (DMPE-mPEG550).
  • An example of a monoglyceride useful as a -fusogen is mono-olein.
  • fusogens may be useful for the invention including but not limited to detergents, calcium salts, sulfates, membrane fusion proteins, membrane glycoproteins, high concentrations of membrane proteins, peptides derived from such fusion and glycoproteins, and fusogenic peptides.
  • the CLB gels of the invention are preferably 30-99% water content, so as to form CLB gels under ambient, protein-friendly conditions. (4) Proteins
  • crystals of proteins are produced by first dissolving the protein to be crystallized in an appropriate aqueous solvent before combining it with the crystallization mixture.
  • aqueous solvents include but are not limited to such agents as detergents, salts, organic solvents, co-factors, or buffer species.
  • the protein is added to the crystallization mixture as a membrane suspension.
  • the CLB gels may be used to crystallize such proteins as membrane proteins, membrane associated proteins, antibodies, lipoproteins, glycoproteins, enzymes, kinases, receptors, channels, and peptides.
  • the CLB gels of the invention may be useful in crystallizing membrane and membrane-associated proteins such as ligand-gated ion channels, G-protein coupled receptors (GPCRs), tyrosine kinase receptors and the tumor necrosis factor receptors.
  • GPCRs G-protein coupled receptors
  • lipid phase additives may be added to the host lipid and fusogen to further promote protein stability and crystallization.
  • Lipid phase additives may include but are not limited to detergents, membrane lipids, and membrane components.
  • Detergents that may be present or added to the CLB gels include but are not limited to, CHAPS series, sodium dodecyl sulfate, monodisperse and polydisperse polyoxy ethylenes (POEs), N-oxide detergents, zwitterionic detergents, peptide detergents, detergents with sugar derivatives as head groups such as octyl glucoside and dodecyl- maltoside, hydroxy alkyl detergents, and bile salts and derivatives such as deoxycholate.
  • Membrane components and dopants that may be used as lipid phase additives may include but are not limited to, lipids, cholesterols, steroids, ergosterols, polyethylene glycols, proteins, peptides, or any other molecules such as fatty acids, triacylglycerols, glycerophospholipids, sphingolipids (i.e.
  • sphingomyelins cerebrosides and gangliosides
  • sterols cholesterol, asymmetric or symmetric bolaamphiphilic lipids, surfactants, polysorbate, octoxynol, diacetylene derivatives, phosphatidylserines, phosphotidylinositols, phosphatidylethanolamines, phosphatidylcholines, phosphatidylglycerols, phosphatidic acid, phosphatidylmethanols, cardiolipins, ceramides, lysophosphatidylcholines, D- erythrosphingosines, sphingomyelins, dodecyl phosphocholine, N-biotinyl phosphatidyl ethanolamine, and other synthetic or natural components of cell membranes that can be associated with a membrane or membrane proteins.
  • the CLB gels can be combined or overlayed with a crystallization solution.
  • the crystallization solution is generally comprised of salts, osmolytes and precipitants.
  • Preferred crystallization solution components may include but are not limited to all buffer components in commercially available crystallization solutions from Hampton (Hampton, Aliso Viejo, CA), Sigma- Aldrich family of chemical companies (Sigma, St. Louis, MO), Nextal Biotechnologies (Montreal, Quebec Canada) and Emerald Biosciences, now owned by Decode Genetics (Reykjavik, Iceland).
  • One or more osmolytes can be added to the crystallization solution in order to promote the stabilization of the diacyl-lipid CLB gels.
  • Some crystallization solutions are already compatible with the gels because of their intrinsically high osmotic strength (see results shown in FIG. 6).
  • crystallization solutions containing osmolytes such as glycerol, inositol, trehalose, or polyethylene glycol in several molecular weight ranges from 100 to 5000 daltons, malonate, high concentrations of phosphate, and methylcellulose can be added and should minimally impact the effectiveness of the basic crystallization solution, yet will serve the purpose of preventing excessive swelling of the CLB gel. (7) Methods of crystallization
  • the method of crystallization using the CLB gels is as follows.
  • the dry host lipid and fusogen are hydrated with an appropriate volume ratio of protein which is dissolved or suspended in water or other buffers and detergents.
  • the host lipid, fusogen and protein are preferably combined using the syringe method described by syringe-based mixing technique as described by Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. (1998) A simple mechanical mixer for small viscous lipid-containing samples, Chem & Phy s Lipids. 95, 11-21.
  • the CLB gel is formed.
  • the CLB gel is preferably then aliquoted in small volumes into a container.
  • the term “container” is meant to include any container suitable for performing the method of crystallization including multi-well containers, microplates, depression wells, test tubes, and any other such container. By “container” it is also meant to include any additional components such as a cover slip.
  • the term “cover slip” is meant to include cover slips as is generally known in the art, cover-slip-like materials, films, or any material used to physically or mechanically hold the gel in a manner suitable for crystallization and/or viewing or detection. Crystallization solutions to promote or allow crystallization are usually added to each gel aliquot. The gels are then allowed various extended periods of time for protein crystals to form. It is contemplated that the CLB gels can be allowed to sit for up to several days, weeks or months to promote protein crystallization.
  • the crystallization solution might be added in the syringe during the lipid mixing/hydration step instead of later in a microwell. Furthermore, it is contemplated that the crystallization solution might be introduced to the lipid before OR after the protein. Ability to visualize crystals is a key parameter for gel formulation and screening.
  • micro-well plates are used having a flat-bottom geometry that provides optical quality that is superior to that of most crystallization trays that are used for soluble proteins.
  • glass bottom microplates (Whatman, Clifton, NJ) allow one to easily use polarization optics as well as dark field or normal bright field optics to monitor the process of crystallization.
  • a gentle flattening of the CLB gel after it is dispensed into a microplate further improves the optical quality by eliminating surface irregularities of the gel.
  • the flattening of the gel can be carried out by centrifugation.
  • flattening of the gel can be carried out with a cover-slip. The coverslips are used to prevent the gel from floating in the crystallization solution, to prevent overhydration, and improve optical quality.
  • a method of flattening such as centrifugation may be used to flatten the gel and a coverslip may not be required.
  • the high optical quality of the preferred setup also allows one to use both polarization optics and dark field optics for examination of gels for crystal formation.
  • Examples of polarization-optical images of both colored and uncolored protein crystals in monoglyceride gels have been published by Rouhani S, Facciotti MT, Woodcock G, Cheung V, Cunningham C, Nguyen D, Rad B, Lin CT, Lunde CS, Glaeser RM, "Crystallization of membrane proteins from media composed of connected-bilayer gels," Biopolymers, 2002; 66(5):300-16 and Misquitta Y, Caffrey M, "Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization," Biophys J.
  • the dimensions of parameter space can be further extended by the addition of lipids from the protein's native membrane as a lipid phase additive.
  • the addition of native membrane lipids in increasing molar ratios may help to find the optimum amount that is available to be recruited as "mortar” between the protein "bricks” (as was found to be necessary for sensory rhodopsin crystallization (Luecke, H., Schobert, B., Lanyi, J. K. & Spudich, E. N. (2001) Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction, Science.
  • Protein crystals obtained by this method tend to be smaller than their soluble protein counterparts. Microcrystals in the size range of 20-30 ⁇ m, which are needed to at least screen diffraction quality, can be unambiguously seen w ⁇ ien removed from the CLB gel and mounted in standard loops and frozen, as is routinely done in the art.
  • Obtaining high quality data from 20-75 ⁇ m crystals is also routine when using a synchotron x-ray source such as beamline 8.3.1 at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory, Berkeley, CA.
  • This beamline has a 30 ⁇ m pinhole collimator, and an advanced crystal positioning system, which, negates most small crystal issues.
  • a 5 x 20 ⁇ m hexagonal crystalline plate of wild-type bacteriorhodopsin grown in the PE CLB system diffracted to 1.8 A on this beamline.
  • the stability assay is a functional assay specific to the protein of interest. Examples of such stability or functional assays which may be used have been described by Schoneberg T, Liu J, Wess J. (1995), Plasma membrane localization and functional rescue of truncated forms of a G protein-coupled receptor, J Biol Chem. 270(30):18000-6; Annemieke van Dalen, Sander Hegger, J.
  • the stability of protein molecules in the CLB gels is evaluated by using an intrinsic fluorescence assay to test the retention of the native protein structure.
  • an intrinsic fluorescence assay to test the retention of the native protein structure.
  • Fluorescence can be used to measure a membrane protein's structural integrity in the various environments required for crystallography, including solubilized protein, protein inserted into the bilayer gel matrix, and protein exposed to crystal-forming precipitant solutions. Therefore a facile method for using trie intrinsic fluorescence of tryptophan residues to monitor whether severe denaturation has occurred in a reconstituted protein should be used. This method can be used to increase the protein crystallization rate of success and to narrow down the set of crystallization mixtures and conditions which will best promote crystal formation of a specific protein.
  • the intrinsic fluorescence assay can be performed as herein described.
  • the intrinsic fluorescence assay works by assessing a protein's susceptibility to thermal denaturation during reconstitution in a CLB gel.
  • several crystallization mixtures of CLB gel are combined with the protein in order to determine which formulation provides the most stable CLB gel to maintain protein structural integrity and function.
  • the different formulations can be performed in a multi-well format, which is well suited for high-throughput screening of a large number of crystallization mixtures and conditions.
  • the intrinsic tryptophan fluorescence spectrum of the protein is measured before and then again after heating the sample to denaturing temperatures, e.g., 90° C for 5 minutes.
  • the intrinsic tryptophan fluorescence spectrum of a protein is a concept known in the art, and can generally be measured as known in the art by excitation of a. protein at about 280 nm, and observing the fluorescence emission spectra of the protein at 300-400 nm.
  • excitation could occur at wavelengths from 250-295 nm, but with the peak generally around 280 nm.
  • the intrinsic tryptophan fluorescence spectra of a protein changes after heating of the protein reconstituted in CLB gel, as compared to before heating, this indicates that the not limited to, changes in intensity (which is an indirect measure of quantum efficiency and/or fluorescence lifetime), waveform changes and ⁇ max shifts.
  • the assay is well-suited to give semi-quantitative comparisons of the stability of a membrane protein in detergent versus in a lipid-bilayer gel, or for that matter in different gel formulations and under different buffer conditions.
  • an excellent correlation was obtained between the result of the intrinsic fluorescence assay and the measurement of the visible absorption spectrum as a way of monitoring protein stability (or denaturation) after the protein was reconstituted into the mono-olein gel. Since the measurement compares the two spectra obtained from the same well, it is robustly immune to the large number of factors that can give well-to-well and day- to-day variations in the fluorescence intensity.
  • performing the intrinsic fluorescence assay should find use in assessing any membrane protein or membrane-associated protein's stability in a CLB gel.
  • FIG. 2 shows a photograph of the hardware components that were used. One syringe is loaded with aqueous buffer and the other with anhydrous lipid, which may be dried in situ from organic solvent.
  • the two syringes are then coupled and their contents are forced back and forth until uniform mixing is achieved.
  • a detergent- solubilized protein is included in the buffer syringe, the membrane protein is spontaneously reconstituted into the lipid bilayers that form as the lipid is hydrated.
  • the detergent disperses (by dilution) into the large-excess reservoir of lipid bilayers. Hydration of the lipid is typically completed within a period of 30 minutes at room temperature.
  • a further advantage of the syringe-based technique is the fact that the Hamilton- syringe ratchet can be used to dispense reproducible aliquots of gel into microplate wells. These aliquots can be as small as 0.2 ⁇ l.
  • the dispensed gel then can be overlaid with crystallization screening buffer and the container sealed with Clear Seal tape (Hampton, Aliso Viejo, CA).
  • a peak at -550 nm represents a slightly perturbed protein in the (dark-adapted) ground state
  • a peak at -410 nm represents a species with a deprotonated Schiff base
  • a peak at -380 ran represents free retinal, indicating denatured protein since the retinal is no longer coupled to the active site.
  • FIG. 3 shows a the absorption spectrum of bacteriorhodopsin (bR) in three different environments: native purple membrane, detergent-solubilized (octylglucoside), and reconstituted into monoolein (MO).
  • Purple membranes exhibit a ⁇ max at 568 nm while bR which is detergent-solubilized or incorporated into MO exhibits a peak shifted to 560 nm or lower. Additionally, when in MO, the peak is considerably broadened toward shorter wavelengths and there is an additional peak at 410 nm, implying the presence of a species with a deprotonated Schiff base.
  • bR has an additional peak at 380 nm which is due to free retinal that is released from denatured protein. Essentially all of the protein is denatured (retinal is released) when the protein is heated while in the MO gel. Thus it was concluded that mono-olein does not stabilize bR as previously thought, and instead exerts a destabilizing influence.
  • Phosphatidylethanolamine (PE) and derivatives of PE represent a class of host lipids that formed optically clear gels when hydrated at a water content roughly in the range from 30% to 99%, depending on the type of lipid. Depending on the type of PE lipid, some were even stable at ⁇ 4 °C, unlike the monoglyceride gels of Example 1 and 2. The first challenge in exploiting these bilayer gels, however, was the need to discover a way to hydrate the lipids and form gels that are protein-friendly.
  • FIG. 4 An example of an x-ray diffraction pattern for such a gel is shown in FIG. 4.
  • This gel was produced using di-18:l-mono-methyl-PE as host lipid, the fusogen used was DMPE- mPEG550. Hydration percentage of the gel was 90% and the ratio of host-lipid:fusogen was 85%: 15%.
  • Crystals of bacteriorhodopsin were obtained with a range of host lipids and fusogenic lipids. Crystals of wild-type bacteriorhodopsin grown in a gel formed comprised with 95% di-18:l-mono-methyl-PE as host lipid, 5% DMPE-mPEG550 as fusogen with 67% total hydration diffracted to 1.8 A resolution. Wildtype bR crystals were observed forming in both the vesiculated (blebby, turbid) periphery as well as in the optically transparent interior of a PE gel formed using 85% di-18:l-mono-methyl-PE as host lipid, 15% MO as fusogen and 67% hydration. The vesiculated periphery is an optically terrible environment, yet crystals of bR remain nearly as easy to detect in this environment as they are in the optically clear gel, because they are strongly colored.
  • FIG. 5A and FIG. 5B show absorption spectra of wt bR and the D96G/F171C/F219L triple mutant respectively, after reconstitution into a gel of di-18:l-monomethyl-PE, 5-20% DOPE-mPEG550 as the fusogenic lipid.
  • the CLB gel was made as described in Example 3.
  • the triple mutant of bR was used in this case because it is a labile mutant.
  • a facile method was used to detect the intrinsic fluorescence of tryptophan residues to monitor whether severe denaturation has occurred in a reconstituted protein.
  • the assay works by assessing a protein's susceptibility to thermal denaturation. It is performed in a 384-well format for high-throughput screening of a large number of crystallization conditions.
  • the intrinsic fluorescence spectrum of the protein e.g. 280 nm excitation, 300- 400 nm emission
  • the intrinsic fluorescence spectrum of the protein e.g. 280 nm excitation, 300- 400 nm emission
  • the result is scored by saying that the protein was already denatured before the sample was heated.
  • the intrinsic fluorescence assay for retention of native structure also confirms, as expected, that native structure is retained in the PE-based gel.
  • FIG. 5C shows that the intrinsic fluorescence of the triple mutant is hardly changed after heating when the protein is in the mono-olein gel, demonstrating that it was already denatured before heating.
  • FIG. 5D demonstrates that there is a large change in the fluorescence after heating when the triple mutant is reconstituted into the PE-lipid gel.
  • the visible spectrum (FIG. 5B) also demonstrates that denaturation occurs as a result of heating.
  • the graphs show that protein is hardly changed after heating when in monoolein, demonstrating that it was already denatured before heating. In contrast, a large change is observed after heating when the protein was reconstituted in PE gel, demonstrating that in this case the protein was sensitive to heating and was therefore initially in a more native-like state.
  • the percentage indicated in the figure refers to the ratio of the fluorescence intensities of the same sample measured before and after heating, while the wavelength value refers to the amount of shift, if any, in the position of ⁇ max.
  • membrane proteins in general, retain their native structure when reconstituted into the PE CLB gels. This can be further verified for non-colored proteins by use of optimal lipid formulations tailored to individual membrane proteins.
  • Optical imaging and formation of protein crystals in the diacyl-lipid CLB gels was enhanced by flattening or squashing an aliquot of gel after it was dispensed into a multi-well glass bottom plate.
  • the flattening of the gel was carried out by centrifugation or with a round
  • 5mm diameter coverslip (Bellco Biotechnology, Vineland, NJ). Use of a coverslip was found to prevent the gel from floating, optical quality was improved and it prevented overhydration.
  • Certain PE formulations were found to remain stable at temperatures lower than room temperature such as 4 °C.
  • CLB gels formed with mono-olein change phase at temperatures below 19 0 C.
  • low temperature gels include, but are not limited to, gels formed with di-18:l-PE as host lipid plus 5-20% DOPE-mPEG350 as the fusogenic lipid. Low temperatures can be favorable for protein stability.

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Abstract

L'invention porte sur des compositions à base de gels bicouches lipidiques assemblés en trois dimensions et sur leurs procédés de préparation et d'utilisation dans la cristallisation de protéines membranaires. Les gels bicouches lipidiques assemblés en trois dimensions sont formés par un mélange de cristallisation comprenant une solution aqueuse et une phase lipidique, la phase lipidique comprenant un liquide hôte et un agent fusogène. Par exemple, on a obtenu des gels stables bicouches lipidiques assemblés en trois dimensions en utilisant un mélange de cristallisation comprenant des lipides hôtes, tels que des phosphatidyléthanolamines, et un agent fusogène, tel que des lipides conjugués à des méthoxy-polyéthylèneglycols.
PCT/US2005/034098 2004-09-24 2005-09-22 Cristallisation de proteines membranaires dans des gels bicouches lipidiques assembles en trois dimensions WO2006036772A2 (fr)

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JP2011500825A (ja) * 2007-10-22 2011-01-06 ザ スクリプス リサーチ インスティチュート 膜タンパク質の高分解能結晶を得るための方法および組成物
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JP2016117739A (ja) * 2008-09-30 2016-06-30 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 結晶化デバイスに負荷する方法
CN102170946A (zh) * 2008-09-30 2011-08-31 恰根有限公司 加载结晶装置的方法
JP2012504152A (ja) * 2008-09-30 2012-02-16 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 結晶化デバイスに負荷する方法
WO2010037510A1 (fr) 2008-09-30 2010-04-08 Qiagen Gmbh Procédé de chargement d'un dispositif de cristallisation
US9352248B2 (en) 2008-09-30 2016-05-31 Qiagen Gmbh Method of loading a crystallization device
EP2168646A1 (fr) * 2008-09-30 2010-03-31 Qiagen GmbH Procédé de chargement d'un dispositif de cristallisation
US20160318974A1 (en) * 2008-09-30 2016-11-03 Qiagen Gmbh Method of loading a crystallization device
KR101762963B1 (ko) * 2008-09-30 2017-07-28 키아겐 게엠베하 결정화 장치의 적재 방법
US10227378B2 (en) 2008-09-30 2019-03-12 Cube Biotech Gmbh Method of loading a crystallization device
US8536306B2 (en) 2008-10-01 2013-09-17 The Scripps Research Institute Human A2A adenosine receptor crystals and uses thereof
EP2832741A1 (fr) * 2013-07-30 2015-02-04 Commissariat A L'energie Atomique Et Aux Energies Alternatives Procédé de cristallisation de protéines membranaires et sur des compositions, dispositifs et kits pour réaliser un tel procédé
RU2819207C2 (ru) * 2020-12-17 2024-05-15 Федеральное государственное бюджетное учреждение науки Институт белка Российской академии наук (ИБ РАН) Способ кристаллизации мембранных белков в липидной мезофазе для поточной кристаллографии

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