Methods for Evaluating Membrane Protein Oligomerization

1. 3-12% gel 1048 720 480 242 146 66 20 1236 1048 720 480 242 146 66 MW 1.0 ml 0.5 ml Bor1p monomer Detergent micelle peak PGAL PGK15’ LIC site ORF LIC site 3C His10 PAHD2 PGAL PGK15’ PGK15’ LIC site LIC site ORF ORF LIC site LIC site 3C 3C ZZ ZZ His10 His10 Bor1p (uncleaved) Bor1p (cleaved) Fraction # 3C-GST Purification Strategy Gel Filtration Superdex 200 Ynl275w • 1. Yeast growth during expression in YPD with autoinduction (GAL1 or ADH2 promoter) in • protease-deficient yeast strains. • 2. Cell lysis by Avestin C3 homogenizer. • 3. Membrane pellet isolation and solubilization in detergent. • 4. Affinity purification by IMAC and/or IgG resins and 3C rhinovirus protease cleavage from resins. • 5. Size exclusion chromatography (SEC) purification to remove aggregates. • 6. Concentration of protein with detergent removal by cycles of dilution and concentration. Talon Elution 1 Talon Elution 2 Talon Elution 3 Urea/SDS stripped Talon IgG super rebound to IgG IgG Elution 1 IgG Elution 2 IgG Elution 3 IgG stripped urea/SDS Elutions after GST resin Marker Bor1p 10 l Bor1p 5 l 3C-GST 5 g Bor1p Crystals from Bor1p purified in DDM Crystals purified in C12E8 with anion channel inhibitor Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology Kathleen Clark*, Nadia Fedoriw*, Joan Randles*, Mary Rosenblum‡, Michael G. Malkowski‡, George T. DeTitta‡, and Mark E. Dumont†* *Department of Pediatrics and †Department of Biochemistry and Biophysics University of Rochester Medical Center Rochester, NY 14642 and ‡The Hauptman-Woodward Institute, 700 Ellicott Street, Buffalo, New York 14203 Abstract Compared to the large amount of structural information available for soluble proteins, transmembrane proteins (TMPs), and particularly TMPs from eukaryotes, remain poorly characterized, despite their physiological and medical importance. The Center for High-Throughput Structural Biology is working to develop improved protocols for expression and purification of TMPs in the yeast Sacchromyces cerevisiae. We have focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays for determining whether the protein is maintained in a native state. Initial purifications were based on expression of the genes under control of a galactose inducible promoter, but higher cell densities and improved expression have recently been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification- some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. We have taken several steps that have effectively reduced proteolysis by endogenous yeast proteases. Size exclusion chromatography in conjunction with static light scattering, refractive index detection, and UV absorption, is being used to characterize the oligomeric state, heterogeneity, and amount of bound detergent in purified protein preparations. The results of these analyses are also being compared with native gel electrophoresis and with colorimetric assays for determining detergent concentrations. Approaches that we have developed for exchanging from an initial detergent that efficiently solubilizes TMPs to alternative detergents more amenable to crystallizations, as well as procedures for efficiently removing excess detergent from purified protein-detergent complexes, appear to be important for successful crystallization. Methods for Evaluating Membrane Protein Oligomerization • Progress Summary • Protein expression – • High-purity yeast transmembrane proteins are now being produced for crystallization. Utilizing the ADH2 promoter over the GAL1 promoter has increased the protein yield of Bor1p by 2X and reduced cost of expression by eliminating galactose as inducer. Expression testing of other proteins using this promoter are in progress. The best yields of purified membrane protein are 0.6 mg/l of culture. • To eliminate the problem of expression plasmid loss during growth in both rich and selective media, we are testing an integrating vector for stable integration into the dispersed Ty δ sites of S. cerevisiase as in Parekh et. al. (1996). • To further reduce protease activity in the yeast lysates, we are testing an additional protease deficient strain BJ1268 (pep4-, prb1-, plus prc1- deficient). Also, we have determined that the best protease inhibitors for yeast proteins are PMSF and chymostatin over Peflaboc SC™ (Roche). • 2.Protein/Detergent Analysis- • Techniques for monitoring protein oligomerization, eg. SEC with LS,UV, and RI and blue native electrophoresis, have been implemented and are being used to monitor protein oligomerization during detergent removal and after adding ligands, inhibitors, and other additivies. • Colorimetric detergent assay has been implemented for analysis of DDM levels in protein samples. SEC with three detector system is being developed for detergent analysis. • Challenges • The goal of “E. coli-fying” yeast as an expression system for membrane proteins will benefit from ongoing development in the following areas: • Increasing native protein production by continued development of culture and induction conditions. • Optimization of detergent selection for solubilization, purification, and crystallization of proteins. • Development of purification protocols that do not rely on cleavage of tags or engineering of specific proteases with enhanced activity toward detergent-solubilized proteins. • Development of a inexpensive high-affinity resin for affinity purifications. • Expansion of expression and purification techniques to include a wider range of targets refractory to current methods. Protein/Detergent Interactions SEC with LS, UV, RI Detection Blue Native Gel (Sample required >10 ug protein) ( >5 ug protein) • Excess detergent in membrane protein preparations • may interfere with crystal contacts formation during • crystallography. • Removal of detergent may cause proteins to aggregate. • Size Exclusion Chromatography with detection by UV absorbance (UV), refractive index (RI), and static light scattering (LS) detection can be used to monitor protein oligomerization. With knowledge of protein extinction coefficients and the instrument calibration constant, the protein mass can be determined independent of hydrodynamic radius and without interference from protein posttranslational modifications, eg. glycosylation, and bound detergent. Additionally, the method can be used to estimate the amount of detergent bound to the protein. (Folta-Stogniew, 2006). • Blue Native Electrophoresis (NativePAGE) can also be • used to monitor protein oligomerization. Coomassie dye re- • places the detergent around the protein preventing aggregation during electrophoresis. The MWs obtained are not accurate but can • be calibrated against other methods. (Heuberger, et. al., 2002). A Before detergent reduction 4-16% Gel mV Time (min) B After detergent reduction Yeast Membrane Proteins Expressed in Yeast 1. To date, eight structures of different heterologously expressed eukaryotic transmembrane proteins have been solved by x-ray crystallography. Five of these proteins were expressed in yeast. 2. Advantages of homologous expression system for post-translational modifications, membrane targeting, protein folding, lipid requirements. 3. Extensive annotation of yeast genome as far as protein-protein interactions, subcellular localization, expression levels, protein function. 4. Availability of yeast strains with altered protein degradation, unfolded protein response, post-translational modifications, intracellular trafficking. 5. Rapid and inexpensive conditions for culturing yeast cells. Reduced detergent micelle peak mV Bor1 monomer Accurate Molecular Mass Determination using Static Light Scattering Time (min) NativePAGE Gel for Panel B SEC of Bor1p in DDM detergent.Panel A: Bor1p before detergent removal. Panel B: Bor1p after detergent removal. In DDM Bor1p is a monomer. Note change in area of micelle peak. C Vectors for yeast membrane protein expression Multi-step purification of the S. cerevisiae boron transporterBor1p Before detergent reduction pSGP36 (Ligation independent cloning) Micelle peak GAL1 promoter - Induction with galactose after glucose depletion mV Detergent: dodecyl maltoside (DDM) Culture: 96,000 ODmls pSGP40 (Ligation independent cloning) Bor1 MW: monomer 65 kDa, dimer = 130 kDa, trimer = 195 kDa pSGP46 (Ligation independent cloning) Calculation of Detergent Bound to Protein ADH2 promoter - Glucose repressible promoter D After detergent reduction mV Time (min) 1. Calculated based on DDM concentrations determined by colorimetric analysis. (DDM concentration in peak maximum- DDM concentration at peak minimum). 2. Calculated using three detector method. 3. Calculated based DDM concentration determined by colorimetric analysis of purified protein and total protein concentration. 4. No detergent assay available for C12E8. Blue native gel for Panel D SEC of Bor1p in C12E8 detergent. Panel C: Bor1p before detergent removal. Panel D: Bor1p after detergent removal. Note increase in protein oligomerization and loss of micelle peak. Determining and Reducing Detergent Concentrations In Bor1p Purifications Detergent Analysis of Bor1p Concentrator Filtrates RI Analysis of Bor1p Concentrates 1048 720 480 242 146 66 20 Using the ADH2 Promoter Increases Protein Expression for Bor1p F A 20X 30X 40X 50X 10X 20X 30X 40X 50X B Bor1p dimer Induction Time (hrs) Ratio RI Signal Area micelle/Area protein The ADH2 promoter is a glucose repressible promoter (Price, 1997). For expression, the yeast cells are grown in rich media. Expression is initiated, after glucose is consumed. For Bor1p under the GAL1 promoter optimal expression occurs at around 12. hrs then decreases. For the ADH2 promoter optimal expression is reached at around 20 hrs. when cell densities are higher. % Dodecylmaltoside Detergent mV 0 6* 24 30 12* IgG Concentrator Filtrate Dilutions Time (min) Concentrator Filtrate Dilutions Panel B: Using SEC with UV, RI, LS detection (see right panel), detergent levels can be evaluated based on the ratio of micelle peak to protein peak area. Note the increase of the area ratio at dilution 5, indicating protein loss at lower DDM concentrations. This SEC method can be a sensitive indicator of protein stability. Panel A: Detergent (DDM) is removed from the purified protein using repeated cycles of dilution and concentration (Millipore 50 kDa concentrator).DDM was analyzed using a colorimetric method (Urbani and Warne, 2004). The DDM concentration of the diluting buffer is 10X lower than the critical micelle concentration (buffer DDM = 0.001%). SEC of Bor1p in C12E8 detergent bound to inhibitor. In C12E8 Bor1p is a preferentially a dimer. Compare the oligomerization state of Bor1p in panels C and D with F. SEC is a fast method for evaluating the oligomerization of proteins. *300 OD mls Gal1 promoter Blue native gel for Panel F ADH2 Promoter Panel C: SDS Page analysis of Bor1p at the different stages of detergent removal. Bor1p Dilution 10X 20X 30X 40X 50X