Analysis of the size and shape of protein complexes from yeast

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step. We have no information on whether the activation was autocatalytic or due to the activity of another protease. Nevertheless, it seems clear that strains of the pep4 PRB1 genotype that contain PrB precursor at levels comparable to levels of PrB in wild-type strains may not be ideal starting strains for biochemical analyses and/or purifications, as the PrB precursor constitutes a potential reservoir for the production of PrB during a purification. This provides an additional reason for using prbl pep4 double mutants.

[8] A n a l y s i s

of the Size and Shape Complexes from Yeast

of Protein

B y SCOTT C. SCHUYLER a n d DAVID PELLMAN

Introduction The era of gene discovery in budding yeast ended a few years ago with the complete sequence of the yeast genome.l Use of the genome sequence and all of the remarkable tools generated from it are now the common currency of everyday life in yeast laboratories. The idea that the genome sequence would change the way we do genetics was easy to anticipate. What was perhaps less obvious was the degree to which the genome sequence would revolutionize biochemical experiments. New methods in mass spectrometry and informatics, now combined with many novel methods for epitope tagging, affinity chromatography, and high-throughput proteomics, have created a boom industry in the discovery and characterization of protein complexes. There is a proud history of biochemistry in yeast dating back to the early studies on metabolism. Because of a new emphasis on analyzing protein complexes, many of the "classic" biochemical approaches have come to the fore. These methods, especially when used in combination with more recently developed procedures, are powerful experimental tools for analyzing the physical properties of protein complexes. This chapter focuses on methods for estimating the molecular weight and shape of soluble protein complexes. We review in practical terms the preparation of native extracts, size-exclusion chromatography (gel filtration), and velocity sedimentation (sucrose gradients). We also briefly comment on commonly used methods to detect protein-protein interactions such as polymerase chain reaction (PCR)-based epitope tagging of proteins followed by coimmunoprecipitation. We anticipate an increasingly wide use of these methods by the community of yeast researchers. 1 A. Goffeau, B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon, H. Feldmann, E Galibert, J. D. Hoheisel, C. Jacq, M. Johnston et al., Science 274, 546 (1996).

METHODSIN ENZYMOLOGY,VOL.351

Copyright2002,ElsevierScience(USA). Allrightsreserved. 0076-6879/02$35.00

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It is worthwhile saying a few words about the circumstances in which yeast researchers will find a need to use these methods. In laboratories where protein complexes are being purified based on a biochemical activity, the protocols described here will be of use to beginning students. However, many experiments with yeast start with the identification of a protein through a genetic screen, by homology, or by an interesting gene expression pattern. In this case, one of the first issues to arise is whether the protein of interest acts alone or as part of a macromolecular complex. The answer to this question can have an important impact on how one interprets genetic data. For example, a protein identified by genetic interactions with tubulin would be viewed differently if it were found to be a monomeric microtubulebinding protein versus being part of a large motor protein complex. One simple and information-rich experiment is to compare the size of pure recombinant protein with that of the protein from a native yeast extract. A second issue is that many proteins have multiple functions and are components of distinct protein complexes. The methods reviewed in this chapter can be used to dissect the partitioning of proteins into different complexes. These methods become particularly informative when used in combination with mutational studies. A third issue is that the discovery of many new protein complexes will invariably lead to studies on the structure of these complexes. The methods described here are critical for the preparation of proteins for structure determination. One benchmark for the suitability of a protein preparation for crystallization is whether it runs as a single discrete peak by sizeexclusion chromatography. Finally, many protein complexes are dynamic, and the ability to determine the size of the complex under different physiologic conditions is important for understanding many aspects of cell signaling and protein regulation. Native Yeast Extracts Here we review two different methods for making native yeast extracts. The N2(liquid) method tends to be more convenient for large-scale extract preparations. Glass bead lysis is quicker and can be used on a small scale, which is more convenient when preparing a large number of samples. For most uses, it is important to keep the extracts concentrated and to avoid proteolysis by use of protease inhibitors (see later). In order to preserve the integrity of protein complexes, it is important to use lysis solutions that are well buffered for pH and do not contain too much salt or detergent.

Liquid Nitrogen Lysis In this protocol, cells in lysis buffer are rapidly frozen in N2(liquid) and can then be stored for an extended period at -80o. 2 In the following method, a low 2 K. B. Kaplan and E K. Sorger, in "Protein Function:A PracticalApproach"(T. Creighton, ed.). IRL Press, Oxford, 1996.

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salt buffer, which should maintain protein-protein interactions, is used for lysis. A typical yield is 3-5 ml of extract at a concentration of 5-15 mg/ml. 1. Grow cells (500-1000 ml) to an OD600 of 0.2-0.5. Harvest cells by centrifugation for 10 min at 4 ° at 13,000g [e.g., JA-14 rotor at 9500 rpm (Beckman Instruments, Palo Alto, CA)]. Pour off the supernatant. 2. Wash cell pellets once in 50 ml of 50 mM HEPES-NaOH (pH 7.4), 150 mM NaC1. This removes excess medium and ensures that the pH is near 7.4. 3. Resuspend cell pellets in an equal volume of lysis buffer [50 mM HEPESNaOH (pH 7.4), 150 m M NaC1, 1 m M phenylmethylsulfonyl fluoride (PMSF), lx protease inhibitors mix]. We use PMSF combined with protease inhibitors such as the "Complete Mini-EDTA Free" protease inhibitor mix (Roche, Indianapolis, IN). A stock of 100 mM PMSF should be made fresh in 100% ethanol. When resuspending the cell pellet, slurries of less than an equal volume do not yield good lysis. Conversely, do not make the extracts too dilute by adding too much lysis buffer. An equal volume mix usually yields a 10-mg/ml extract. Detergents, salts, inhibitors, and other small molecules should be included if necessary (see later). Freeze the cell slurry by dropping the cell suspension from the tip of a pipette directly into N2(liquid) drop by drop and store at - 8 0 ° until use. The slurry forms little frozen beads in the N2(liquid). Do not thaw and refreeze the mix. 4. Grind frozen cell beads with a mortar and pestle (50-100 strokes) into a fine powder that is kept cold in Na(liquid). It is important to precool and keep the mortar, pestle, and a spatula cool. For 0.5-1 liter cultures, grind by hand in a mortar/pestle (Coors, Golden, CO); for larger volumes, grind the cells using a stainless-steel blender chilled with N2(liquid). Do not use ball-beating blenders because Nz(liquid) can freeze and crack the ball bearings. Use carbon-brushed blenders such as those made by Waring (see Fischer, Pittsburgh, PA). One can check the lysis efficiency by looking at the cells in a microscope. 5. Collect powder with a cold spatula into a 15-ml conical tube and spin for 15 min at 4 ° at 2000g to concentrate [e.g., an RT-6000D tabletop centrifuge at 3000 rpm (Sorvall, Newtown, CT)]. Transfer the whole lysate and thick pellet to 1.5-ml Eppendorf tubes and spin for 10 min at 4 ° at 10,000g to make a low-speed supernatant (e.g., top speed in a microfuge). Transfer the superuatant to a new Eppendorf tube. This is the low-speed extract. 6. To make a high-speed supernatant, spin for 1 hr at 4 ° at 100,000g [e.g., a RP 100 AT3-168 rotor in an RC M 120 miniultracentrifuge at 51,000 rpm (Sorvall) or a TLA 120.1 rotor in an Optima TLX Ultracentrifuge at 50,000 rpm (Beckman Instruments)].

Glass Bead Lysis In this protocol, cells are lysed by vortexing them in the presence of lysis buffer and glass beads. 3 This method is faster than the mortar and pestle N2(liquid) method

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and is easier to use with multiple small samples. Using this method, 1 ml of extract at a concentration of 10-20 mg/ml can routinely be obtained. 1. Grow cells (100-200 ml) to an OD600 of 0.2-0.5. Harvest cells by centrifugation for 10 min at 4 ° at 13,000g [e.g., a JA-14 rotor at 9500 rpm (Beckman Instruments)]. Pour off the supernatant. 2. Resuspend pellet in remaining liquid and transfer to a 2-ml screw cap tube. Make sure these tubes (Fischer, PA) fit into the Mini-BeadBeater (Biospec Products, Bartlesville, OK). If the volume is too big (more than 1.5 ml), split the sample into multiple tubes. 3. Spin down screw cap tubes in an Eppendorf centrifuge for 1 min at 4 ° at 10,000g (l 3,000 rpm). Aspirate away all of the medium. At this point, if you want to freeze down the pellet, wash twice with ice-cold lysis buffer and store at - 8 0 °. To proceed with the lysis, after washing twice with lysis buffer, spin for 1 min at 13,000 rpm (10,000g) at 4 ° and aspirate away lysis buffer. 4. Resuspend the cell pellet in 300 #1 ice-cold lysis buffer + protease inhibitors (see earlier discussion). Add an equal volume of prechilled acid-washed glass beads (Sigma, St. Louis, MO) with a diameter of 4 5 0 0 / z m . Place tubes in an ice-water slurry. 5. Make sure the tubes are tightly capped. In a cold room, set the MiniBeadBeater (Biospec Products) to 20-sec pulses at a speed of"50". Beat for 20 sec and then place the tubes on ice for 1 min to cool down in between pulses of bead beating. Repeat five times so there is a total of 100 sec of beating. One can check the lysis efficiency by looking at the cells in a microscope. 6. To separate extract from beads, use a thin pipette tip. However, we have obtained better recovery using the following centrifuge-based method: (a) cut the cap off an Eppendorf tube, (b) invert the 2-ml screw cap tube that contains the beads and extracts, and tap it a few times to get its contents off the bottom of the screw cap tube, (c) use a needle to poke a hole at the very bottom of the screw cap tube, and (d) put the screw cap tube, sitting right on top of the capless Eppendorf tube, into the 15-ml conical tube so that the Eppendorf tube can collect the extract. Spin the 15-ml tube for 3 min at 4 ° at 2000g [e.g., a tabletop centrifuge at 3000 rpm (Sorvall)]. Carefully remove screw cap tubes with a forceps. Take out the Eppendorf tube with the extract. 7. Transfer extract to a new Eppendorf tube. Spin for 15 min at 4 ° at 10,000g. 8. Transfer the supernatant to a new tube. This is the low-speed extract. 9. To make a high-speed supernatant, spin for 1 hr at 4 ° at 100,000g [e.g., a RP100 AT3-168 rotor in an RC M 120 miniultracentrifuge at 51,000 rpm (Sorvall) or a TLA 120.1 rotor in an Optima TLX Ultracentrifuge at 50,000 rpm (Beckman Instruments)]. 3 E. Harlow and D. Lane, "Using Antibodies:A LaboratoryManual." Cold SpringHarborLaboratory Press, Cold Spring Harbor, NY, 1999.

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Troubleshooting One of the main challenges for biochemical manipulations is maintaining the integrity of the protein complex in extracts. It is important to work at 4 ° and to include protease inhibitors in the lysis buffer. If the protein to be studied is present in very small amounts, then it may be necessary to start with a large number of cells and scale the amount of extract to the absolute amount of the protein. It may also be necessary to include small molecules such as phosphatase inhibitors in order to maintain complex structure. Commonly used phosphatase inhibitors include 1 mM NaVO4 (sodium-orthovanadate), 5 mM sodium-pyrophosphate, 10 mM sodium/~-glycerophosphate, and 10 mM sodium-fluoride. Another issue when making extracts is that of efficient extraction and solubility. Proteins vary dramatically in their solubility from being essentially insoluble (<10 ttg/ml) to very soluble (>300 mg/ml). Key variables that affect the solubility of a protein include pH, ionic strength, temperature, and the polarity of the solvents .4 Once cells are lysed it is important to remove any unlysed ceils or large particles by centrifugation. Making a low-speed supernatant is sufficient most of the time, but the supernatant may still be viscous and contain large particles. It is preferable to use a high-speed spin to clarify the extract; however, this may lead to pelleting of the protein of interest. Increasing the salt or detergent concentrations can promote solubility, but may also disrupt protein complex integrity. Finally, it may be necessary to supplement the lysis buffer with salts (many enzymes bind to metals such as Cu 2+, Zn 2+, Ca 2+, Co 2+, and Ni 2+) and other small molecules (such as ATP) in order to maintain protein-protein interactions. Estimating Protein Complex Molecular Weight and Shape Proteins in yeast vary in molecular mass from ~ 3 to ~470 kDa. Most proteins in yeast have molecular masses in the range of 10-150 kDa. 5 Protein complexes of course may be much larger. For example, the yeast 20S proteasome core particle is composed of 28 proteins with a combined molecular mass of ~670 kDa. 6 The following methods apply to either monomeric proteins or protein complexes. Regardless of molecular size and quaternary structure, proteins and protein complexes have a variety of shapes. Protein shapes range from globular (compact and spherical) to extended (rod shaped). Most proteins tend to be globular, but those containing a-helical coiled-coil motifs commonly form extended rods. 7 Estimates 4 D. R. Marshak (ed.), "Strategies for Protein Purification and Characterization." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1996. 5 M. C. Costanzo, M. E. Crawford, J. E. Hirschman, J. E. Kranz, E Olsen, L. S. Robertson, M. S. Skrzypek, B. R. Braun, K. L. Hopkins, P. Kondu et al., Nucleic Acids Res. 29, 75 (200l). 6 M. Bochtler, L. Ditzel, M. Groll, J. Hartmann, and R. Huber, Annu. Rev. Biophys. Biomol. Strucr 28, 295 (1999). 7 K. Beck and B. Brodsky, J. Struct. Biol. 122, 17 (1998).

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of the molecular weight of a protein and its shape in a native complex are made from measuring its hydrodynamic properties in solution. Two hydrodynamic values that can be determined experimentally from the methods outlined later are the Stokes radius (a) from gel filtration and the sedimentation coefficient (s) from sucrose gradients. The Stokes radius is the radius of a hypothetical sphere that has the same hydrodynamic properties as the protein or protein complex. Stokes radii are usually given in nanometers. The sedimentation coefficient of a protein is equal to the rate of movement of the protein through a centrifuge tube divided by the centrifugal force and reflects the molecular weight and shape of the protein. Sedimentation coefficients are usually given in Svedberg (S) units. One Svedberg is 1 × 10 -13 sec. In addition, the Stokes radius can be correlated with the shape of a protein or complex. In a commonly used approach, one models the protein(s) as a prolate ellipsoid. 8 In a prolate ellipsoid, the up/down ( a 0 and forward/back (a2) axes are small and equal to each other, whereas the left/fight (b) axis is larger (al = a2 < b). A large axial ratio (a : b) suggests that the protein(s) has an extended rod shape.

Gel F i l t r a t i o n Analytical gel filtration is a common experimental method used to measure the Stokes radius of a protein or complex. A gel filtration column contains porous beads, and the movement of a protein into and out of the small pores during a column run is influenced by the size and shape of the protein. This is illustrated by comparing two monomeric proteins of the same molecular weight, where one is spherical and the other is rod shaped. During gel filtration the spherical protein, which has a small Stokes radius, will diffuse more readily into the small pores in the bead matrix and thus explore a larger volume and elute later from the column. A rod-shaped protein has a larger Stokes radius and does not explore as much of the solution volume inside the beads. Relative to the globular protein of the same mass, the protein with a rod shape will appear to be bigger by gel filtration because the protein explores less volume and elutes earlier. In general, there are two kinds of gel filtration columns: preparative and analytical. Large volumes can be loaded onto preparative columns, but the resolution is poor. Analytical columns have high resolution, but work best with small load volumes. Manufacturers usually recommend that one does not load more then 5 % of the total column volume. However, a smaller load volume gives higher resolution, and optimal results are obtained with loads of only 1 or 2% of the total column volume.

8 An ellipsoid is a solid of which all the plane sections normal to one axis are circles and all the other plane sections are ellipses. Prolate means lengthened in the direction of a polar diameter, or growing or extending in width. See Ref. 16.

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TABLE I GLOBULAR MOLECULAR WEIGHT MARKERS FOR CALIBRATING ANALYTICAL GEL FILTRATION COLUMN AND STANDARDS FOR SUCROSE GRADIENTSa

Protein

Stokes radius (nm)

Sedimentation coefficient (S)

M

Thyroglobulin Ferritin Catalase Aldolase Yeast Alcohol Dehydrogenase Bovine serum album Cytochrome c

8.5 6.1 5.2 4.8 4.6 3.5 1.0

19.4

349 kDa × 2 = 698 kDa

11.3

51 kDa × 4 = 206 kDa

7.4 4.3 1.9

37 kDa x 4 = 150 kDa 66 kDa x 1 = 66 kDa 12 kDa × 1 = 12 kDa

a From Pharmacia Biotech and Sigma. Some of the markers are themselves multisubunit protein complexes (values are from the manufacturer).

To determine the Stokes radius, one must first determine a Kd, the distribution coefficient. Kd can be thought of as the percentage of the solution volume inside the beads explored by a protein relative to the total solution volume inside of the beads. Siegel and Monty 9 found that the cube root of Kd, (Kdl/3), is linear with the Stokes radii of proteins. Kd is defined as Kd =

[(Ve - - V o ) / ( V t

-

Vg -

Vo)]

(1)

where Ve is the volume that the sample protein elutes in, Vo is the void volume (the solution volume outside of the beads), Vt is the total volume in the column, and Vg is the volume in the column occupied by the bead matrix. Before an experiment, the column should be equilibrated in a low salt buffer to ensure that protein-protein interactions are not disrupted. An analytical column can be calibrated with globular markers of known Stokes radii (see Table I). Calf thymus DNA can be used to determine the void volume (Vo) of the column, and acetone is used to determine the total solution volume. The volume in the column occupied by the bead matrix (Vg) is determined by subtracting the total column volume (provided by the manufacturer) from the total solution volume (determined by acetone). To generate the linear calibration curve for your column, determine the elufion volume (Ve) for each marker and then plot the Stokes radii of the markers versus their K dl/3. Experimental Protocol Commonly used low-pressure analytical gel filtration columns include Superose 6 and Superose 12 (Pharmacia Biotech, Piscataway, NJ) or SE-1000 (Bio-Rad, Hercules, CA). These columns have different molecular weight ranges, 9 L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta 112, 346 (1996).

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where the molecular weight information provided by the manufacturer is for globular proteins or complexes. If there is no estimate of the size of the protein or complex of interest, one has to try several different columns. These columns are not gravity flow columns and should be used in combination with pumps such as the "FPLC Basic" (Pharmacia Biotech) or "BioLogic HR" (Bio-Rad) systems. One may also use gravity flow columns, which are much less expensive. However, the results may not be as reproducible when compared to pump-driven columns. 1. All work should be done on ice, in a refrigerator, or a cold room at 4 °. 2. Equilibrate the column with 3 column volumes of buffer [e.g., a 50 mM HEPES-NaOH (pH 7.4), 150 mM NaCI solution]. The column running buffer should be matched with the yeast extraction buffer. We typically do not add protease inhibitors to our column running buffer; however, one may wish to add these (i.e., if you find that proteolysis is a problem during the column run) or other small molecules. Be sure to follow the manufacturer's guidelines for flow rate and back pressure limits. 3. Load the sample onto the column, but save a portion of the load for analysis after the column run. Typical load volume limits are 2% of the total column volume. Avoid overloading as it creates broad elution profiles and decreases the overall column resolution. Follow the recommendations made by the manufacturer regarding sample preparations and load limits. Samples should not be too concentrated (70 mg/ml or less) or viscous. Samples should be filtered or spun at a high speed to remove any large particles before they are loaded onto the column. Load high-speed supernatant extracts when possible. When using a filter, save a sample of prefiltered extract and compare it with the filtered extract to check if the protein(s) stuck to the filter. To minimize protein loss during filtration, use a polyvinylidene diftuoride (PVDF)-based 0.45-#m sterile syringe filter that has reduced protein binding (e.g., Millex-HV filters, Millipore, Bedford, MA). 4. After loading, run 1-2 full column volumes of buffer through the column at a slow and continuous flow rate. Use a flow rate of 0.1-0.5 ml/min for a Superose 6 or SE-1000 column. Slower flow rates tend to give better column resolution. 5. Collect fractions during the entire column run for analysis. Collect 0.5- to 1.0-ml fractions from a 15-ml column [e.g., using an automated Model 2128 fraction collector (Bio-Rad)]. 6. Assay the load sample and column fractions for the protein of interest by biochemical activity or Western blotting. The maximum(s) of the peak(s) of biochemical activity should be used as an average value for the elution volume. Using Eq. (1) and the elution volume (Ve), determine the Kd~/3 value for the protein of interest. Then, determine the Stokes radius for the protein based on the standard curve for the column. This Stokes radius value is used for estimating the molecular weight and shape of the protein in solution (see later). Analytical columns should be stable and peak elution volumes should be reproducible. Multiple runs should be performed to ensure reproducibility.

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Troubleshooting Keys to good analytical gel filtration are quality sample preparation and slow and even column runs. The extract preparations outlined earlier should be sufficient for quality sample preparation. One potential problem is that the small sample load volume (~250 #I of extract for a 15-ml column) necessary for good column resolution may lead to a loss of biochemical activity. For example, the activity could be a Western blot signal, which may become too dilute after the column run. As such, it may be necessary to concentrate the fractions before analysis. One can precipitate the fractions [using trichloroacetic acid (TCA), see steps 4-7 in the denaturing lysis protocol outlined later] before loading them onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for Western blot analysis to ensure a robust signal. The goal of the methods outlined for analytical gel filtration and sucrose gradients (see later) is to estimate the molecular weight and shape of a protein complex. These values can be changed in solution by the association of proteins with detergents. Thus, it is best to avoid detergents when preparing yeast extracts for analysis. However, you may find that you get poor extraction in the absence of detergent or poor solubility of the protein complex. In this case, it may be necessary to try extracting at a higher salt concentration or including detergent. If detergent is included, try to use it at the lowest level possible and do not exceed the critical micelle concentration. Sucrose Gradients Sucrose gradients are used to determine sedimentation coefficients. Both the molecular size and the shape of the protein affect how it moves through solution during centrifugation. Massive globular proteins sediment further into the gradient compared to smaller globular proteins. Also, an extended rod-shaped protein has a larger frictional coefficient compared to that of a compact sphere of equal molecular weight. Therefore, a globular protein sediments faster than a protein with an extended rod shape. The movement of a protein or a complex through a sucrose gradient is determined by a combination of several forces. The sedimenting force on a particle is equal to the mass (m) multiplied by the centrifugal field w2r, where co is the angular velocity of the rotor (in radians per second) and r is the radius or distance of the panicle from the axis of rotation. As the panicle travels further away from the axis of rotation, the centrifugal force increases. Opposing the sedimentation force are (i) flotation force, (ii) frictional resistance, and (iii) diffusion. The flotation force is equal to mwZrvp, where v is the partial specific volume (i.e., the volume displaced by 1 g of sedimenting panicles) and p is the density of the solution. The net sedimentation force is equal to [mwZr(1 - vp)]. Frictional resistance against a particle moving through solution is equal to (fv), wherefis the frictional coefficient

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and v is the particle velocity. Finally, diffusion is the result of random thermal motions of particles in all directions and leads to the equal dispersion of particles in solution. Experimental

Protocol

To measure the sedimentation coefficient of a protein or complex, it is standard to use either 5-20% (for proteins or complexes from 1S to 20S) or 10-40% (for proteins or complexes from 1S to greater than 20S) sucrose gradients spun in swinging bucket rotors, which have a constant volume-to-radius ratio. These sucrose gradients are nearly isokinetic, which means that a particle of a given mass moves at a constant velocity through the gradient. The velocity is constant because the increase in centrifugal force, as the particle moves further from the axis of rotation, is balanced by an increase in density and viscosity of the solution. It is also common to use a 5-20% glycerol gradient, which has a very similar density profile as a 5-20% sucrose gradient. Glycerol has the added advantage that many enzyme activities and protein complexes are stabilized in it. During centrifugation the maximal rotor speeds (rpm) and the shortest durations should be used because this minimizes peak broadening by diffusion. This is particularly a concern for 5-20% gradients because they are less viscous and are disrupted more easily by diffusion. It is necessary to have molecular weight standards with known sedimentation coefficients to construct a standard curve (Table I). Always have one separate centrifuge tube for the standards during each experiment. Do not mix the molecular weight standards in with a yeast extract because in vitro interactions may cause the standards and/or the protein(s) to sediment abnormally. As with gel filtration chromatography, it is important to prepare a quality sample. Viscous and particulate extracts should be avoided, as these can lead to streaming effects: particles that are not free to diffuse may be aberrantly dragged down into the gradient by other larger particles. Do not load too much sample onto the gradient, and volume load limits should be followed (Table IIl°). It is also important that the load sample is not too dense or highly concentrated, as this can lead to broadening of the peaks and poor gradient resolution. When the sample density exceeds the initial density in the gradient, mixing effects can occur. Diffusion of large protein particles in a highly concentrated load sample is slowed by particle-particle interactions. The small particles that make up the density gradient, such as sucrose, diffuse and tend to mix rapidly into the less mobile sample and destroy the linearity of the gradient. Use extracts that are at a concentration of 5 mg/ml or less for a 5-20% gradient. l00. W. Griffith, "Techniques of Preparative, Zonal and Continuous Flow Ultracentrifugation." Beckman Instruments Inc., Palo Alto, CA, 1986.

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TABLE II PARAMETERS FOR RUNNING SUCROSE GRADIENTSa

Rotor SW 65 Ti SW 60 Ti SW 55 Ti SW 50.1 SW 41 Ti SW 40

Sample volume (ml)

Gradient density (%)

Revolutions per minute

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.5 0.5 0.5

5-20 10-40 5-20 10-40 10-40 5-20 10-40 5-20 10-40 10-40

65,000 65,000 60,000 60,000 55,000 50,000 50,000 40,000 40,000 40,000

a All are Beckman swinging bucket rotors (Beckman Instruments). Information on recommended sample volume loads can be obtained from the manufacturer (O. W. Griffith, "Techniques of Preparative, Zonal and Continuous Flow Ultracentrifugation." Beckman Instruments Inc., Palo Alto, CA, 1986).

1. All work should be done on ice, in a refrigerator, or a cold room at 4 °. Precool the centrifuge and rotor before spinning. 2. Prepare a stock solution of a high percentage (40-60% w/v) sucrose and filter sterilize it. Measure the refractive index of this stock solution to determine the percentage sucrose accurately (see step 7). Prepare one low (5 or 10) and one high (20 or 40) percent solution, both with a final concentration of 50 mM HEPES-NaOH (pH 7.4) 150 mM NaC1. As with gel filtration, we recommend that the buffer used to make the extracts is the same as the buffer used for pouring the gradients. Again, avoid detergents and high salt solutions. 3. Pour sucrose gradients (5-20% or 10-40%). The total gradient volume depends on the rotor and centrifuge tubes. Follow the manufacturer's recommendation on the type of tube and the maximum volumes allowed for any given tube. Gradients should be poured slowly and carefully to avoid mixing. Use a solution flow rate of 1 ml/min or less. To obtain a high degree of reproducibility from tube to tube and experiment to experiment, use one of the automated dual pump systems, such as the "FPLC Basic" (Pharmacia) or "BioLogic HR" (Bio-Rad). Gradients made by simple gravity flow mixing chambers are less reproducible, although they are much less expensive. Regardless of the device used, the tubing leading into the centrifuge tube should be placed on the side of the centrifuge tube. This allows the drops to roll down the side rather then drip into the center of the centrifuge tube, which would disrupt the gradient. Do not let tubes sit unused for an extended time (more than 2 hr), as diffusion will destroy the gradients.

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4. Load the recommended volume of extract onto the top of the gradient (Table II). Save an aliquot of material as a loading control for analysis after centrifugation. If multiple gradients are to be compared, be sure to load an equal volume on top of each one so that the total solution volume from tube to tube remains the same. This is also true for the molecular weight marker tube. Do not disrupt the gradient. Load the tube with either a pipette tip or a needle resting against the side of the tube at a slow rate, drop by drop. Once the centrifuge tubes are loaded, balance each one with lysis buffer one drop at a time. When prepared properly, the tubes should be nearly balanced to begin with. 5. Spin at 4 ° at or near the top rotor speed. Use the rotor speeds provided by the manufacturer (Table II). Use slow acceleration and deacceleration rates to avoid disrupting the gradient. Although optimal times are determined empirically, anywhere from 6 to 16 hr should be adequate for average samples. Sufficient run times should be used to ensure good separation within the gradient, but extended run times should be avoided as they can lead to broadening of the peaks by diffusion. On inspection after the run, the molecular weight marker tube should have one red band (cytochrome c) near the top and green band (catalase) clearly separated down into the tube. 6. Collect fractions at 4 °. Carefully insert a glass capillary tube connected to a peristaltic pump (Bio-Rad). Collect at a flow rate of 1 ml/min from the bottom of the tube. One can also poke a hole in the bottom of the tube and collect the drops. Either way, be sure to scrape off any pellet at the bottom of the tube, which contains very large complexes that may have quickly sedimented to the bottom. It is convenient to collect 0.5- to 1-ml fractions. 7. Confirm the linearity of the gradients by refractometry following the manufacturer's protocol (#ABBE-3L, Thermo Spectronic, Rochester, NY). Plot fraction number versus percentage sucrose. The plot should show that the gradients are nearly linear. The slopes of the gradients should be the same from one tube to the next. 8. For molecular weight marker fractions it is sufficient to run an SDS-PAGE gel followed by Coomassie staining. Remember that some markers are multisubunit complexes, which are denatured when run through SDS-PAGE gels (Table I). Generate a graph of fraction number versus sedimentation coefficient (in Svedberg units). The slope of this graph should be linear. 9. Assay load sample, pellet, and fractions for the protein of interest. The fraction(s) that contains the maximum(s) of the peak(s) of biochemical activity, such as a signal on a Western blot, should be used as the fraction value(s) when determining the sedimentation coefficient. Using the molecular weight standard curve, estimate the sedimentation coefficient for your protein. This is only an estimate and it is usually reported without any decimal places. Sedimentation coefficient values for standard markers are usually measured under standard conditions of 20 ° and in pure water (s20.w). Although the protocol outlined here recommends using nonstandard conditions, the markers and the complexes in the extracts should be

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affected in the same way by the difference in temperature and density. As such, one can present the measured sedimentation coefficient of a protein as an sz0,w value. Troubleshooting The small sample load volume (200-500 #l of extract) necessary for good gradient resolution may lead to a loss of biochemical activity or Western blot signal. As such, it may be necessary to perform a concentrating step on the fractions before analysis. If the protein of interest is being assayed by Western blotting, you can TCA precipitate the fractions before loading them onto SDS-PAGE gels (see steps 4-7 in the denaturing extract protocol outlined later). Finally, the density of most proteins is between 1.3 and 1.4 g/cm 3. However, proteins containing large amounts of phosphate (phosvitin, 1.8 g/cm 3) or lipid moieties (fl-lipoprotein, 1.03 g/cm 3) are substantially different in density compared to the average protein. This difference in density can affect the calculation of molecular weight 4 (see later). Hydrodynamic Calculations Protein complex molecular weight is estimated using the method of Siegel and Monty. 9 The equation that relates molecular weight (M) with the Stokes radius and sedimentation coefficient is M = [(67rrlNas)/(1

-

vp)]

(2)

where N is Avogadro's number (6.022 × 1023), a is the Stokes radius in centimeters, and s is the sedimentation coefficient (in Svedberg units or 1 x 10 -13 sec). 9 Standard values for the density of water (p = 1 g/cm 3) and buffer viscosity (r/= 0.01005 g/cm sec, or 0.01005 poise) can be used. Water has a viscosity close to that of 0.01 poise, as do solutions that are made mostly of water (i.e., lysis buffers). It should be noted that these commonly used values for density and viscosity are approximations based on the assumption that the experiment is performed under standard conditions of 1 atm of pressure at 20 ° in pure water.l° Here we are outlining a very simple method for estimating the molecular weight and it is sufficient to use these values with the assumption that the nonstandard conditions shall have the same uniform effect on the molecular weight markers and proteins in the extract. The average specific volume (v) of a protein can be estimated using the method of Cohn and Edsall u (see Perkins 12 for a more recent discussion). Calculate the average specific volume using Table III 11.12 by adding up the values for the given u E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides." Reinhold, New York, 1943. 12 S. J. Perkins, Eur. J. Biochem. 157, 169 (1986).

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TABLE III VOLUMES AND PARTIALSPECIFIC VOLUMES(v) OF AMINO AC1DSa Volume Amino acid

x l 0 -3 nm 3

cm3/g or ml/g

lle Phe Val Leu Trp Met Ala Gly Cys Tyr Pro Thr Ser His Glu Asn Gln Asp Lys Arg

168.9 187.9 141.4 168.9 228.5 163.1 87.2 60.6 106.7 192.1 122.4 117.4 91.0 152.4 141.4 117.4 142.4 114.6 174.3 181.3

0.90 0.77 0.86 0.90 0.74 0.75 0.74 0.64 0.63 0.71 0.76 0.70 0.63 0.67 0.66 0.62 0.67 0.60 0.82 0.70

a The overall average partial specific volume for all 20 amino acids is 0.724 cm3/g or an average density of 1.38 g/cm 3 [E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides." Reinhold, New York, 1943; S. J. Perkins, Eur J. Biochem. 157, 169 (1986)].

amino acid sequence in the complex and dividing by the total number of amino acids. If the primary amino acid sequence of the protein or protein complex is not known, use the average value of 0.724 cm3/g. It is important to stress that this method for calculating molecular weight is only an estimate with a typical systematic error of about 4-20% of the calculated value. 1°,13-15 The Stokes radius can be correlated with the shape of a protein or complex after making an assumption about the percentage hydration of the protein(s) and 13 j. Steensgaard, S. Humphries, and S. E Spragg, in "Preparative Centrifugation: A Practical Approach" (D. Rickwood, ed.). IRL Press, Oxford, 1992. 14 M. Potschka, Anal. Biochem. 162, 4 (1987). 15 C. M. Field, O. al-Awar, J. Rosenblatt, M. L. Wong, B. Alberts, and T. J. Mitchison, J. Cell Biol. 133, 605 (1996).

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modeling the protein(s) as a prolate ellipsoid. This does not mean that the complex literally has an ellipsoid shape, but rather that the hydrodynamic behavior of the complex matches the predicted hydrodynamic behavior of an ellipsoid with a given axial ratio. In fact, the geometric shapes of proteins are often quite irregular. Nonetheless, if a protein is found to have a large axial ratio, it suggests a rod-like shape versus a globular shape. To determine the axial ratio, first determine the translational frictional coefficient ( f ) from the measured Stokes radius (a) and then compare it with the translational frictional coefficient (f0) of a perfect sphere with the same molecular weight (M) as your complex13,16: f = 6zrr/a = [M(1 - vp)]/(Ns)]

(3)

f0 -----6zr r/[(3vM)/(4rr N)] 1/3

(4)

( f /fo) = a/[(3vM)/(4zr N)] 1/3

(5)

The P hydrodynamic shape function provides a relationship between the translational frictional coefficient ratio (fifo) and the axial ratio (a : b). Determine the P function value for the protein complex by P = ( f / f o ) [(w/vp) + 1] -U3

(6)

Typical hydration values (to) are 0.35-0.40, and v and p are the same as in Eq. (2). 13'16'17 Table IV contains relationships between the P hydrodynamic shape function value and axial ratios (a:b) when assuming a prolate ellipsoid s h a p e . 13,16

PCR-Based Epitope Tagging We briefly comment on PCR-based epitope tagging of proteins and provide one example of a simple coimmunoprecipitation protocol. Western blotting of an epitope-tagged protein is a simple approach for following the behavior of a protein through the procedures outlined in the previous sections. Coimmunoprecipitation from naive yeast extracts is a standard approach for identifying proteinprotein interactions. 18 Coimmunoprecipitations can also be performed on gel filtration or sucrose gradient fractions to determine if the cofractionation of proteins in fact reflects a complex between the proteins of interest.

16 S. E. Harding and H. Colfen, Anal. Biochem. 228, 131 (1995). 17 A. M. Taylor, J. Boulter, S. E. Harding, H. Colfen, and A. Watts, Biophys. J. 76, 2043 (1999). 18 E. M. Phizicky and S. Fields, Microbiol. Rev. 59, 94 (1995).

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TABLE IV RELATIONSHIPBETWEENP VALUEAND AXIAL RATIOa P value

Axial ratio

1.0000 1.0009 1.0031 1.0063 1.0103 1.0149 1.0201 1.0256 1.0315 1.0377 1.0440 1.1130 1.1830 1.2500 1.3140 1.3750 1.4340 1.4900 1.5430 1.9960 2.3590 2.6710 2.9500 3.2050 3.4420 3.6640 3.8740 4.0740

1.0000 1.1000 1.2000 1.3000 1.4000 1.5000 1.6000 1.7000 1.8000 1.9000 2.0000 3.0000 4.0000 5.0000 6.0000 7.0000 8.0000 9.0000 10.000 20.000 30.000 40.000 50.000 60.000 70.000 80.000 90.000 100.00

a Assuming a prolate ellipsoid of revolution [J. Steensgaard, S. Humphries, and S. P. Spragg, in "Preparative Centrifugation: A Practical Approach" (D. Rickwood, ed.). IRL Press, Oxford, 1992; S. E. Harding and H. Colfen, Anal. Biochem. 228, 131 (1995)].

Several elegant and rapid PCR-based methods take advantage of homologous recombination in yeast to generate a variety of epitope-tagged proteins. Tags can be placed anywhere in an open reading frame (ORF), 19,2° as well as at the NH2 19 B. L. Schneider, W. Seufert, B. Steiner, Q. H. Yang, and A. B. Futcher, Yeast 11, 1265 (1995). 20 M. Knop, K. Siegers, G. Pereira, W. Zachariae, B. Winsor, K. Nasmyth, and E. Schiebel, Yeast 15, 963 (1999).

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and COOH termini. 21-29 Epitope tags are most often placed at the COOH terminus because one-step NHz-terminal tagging methods remove the native promoter. Some of these tagging systems take advantage of selectable markers that are unique and do not share homology with S a c c h a r o m y c e s cerevisiae or S c h i z o s a c c h a r o m y c e s p o m b e genes. This is beneficial because it increases the frequency of the desired recombination event. With the continued identification of novel selectable markers, it is quite likely that several new PCR-based tagging cassettes shall soon become available. 3° Once a gene is tagged, it is necessary to confirm that the protein is expressed and that the tagged protein is functional. The following simple denaturing lysis method is used for rapidly screening through potential recombinant clones or confirming the expression of an epitope-tagged protein. D e n a t u r i n g Lysis

This protocol is a convenient and rapid way to isolate denatured whole cell yeast extracts from small culture volumes for SDS-PAGE and Western blot analysis. 31 1. Grow cells (5 ml) to an OD600 of 0.2-0.5. Harvest the cells by centrifugation for 10 min at 4 ° at 13,000g (i.e., top speed in a microfuge). Resuspend the cell pellet in 600/zl of doubly distilled H20 at 4 °. As an aside, when collecting closely spaced time points, one can skip the water wash and add 100/zl of lysis buffer directly to 600 #1 of cells in medium. 2. Add 100 #1 of lysis buffer (1.85 M NaOH, 7.4% 2-mercaptoethanol). Incubate sample on ice for 15 min. 3. Spin for 10 min at 4 ° at 13,000g (i.e., top speed in a microfuge). 4. Place supernatant, which should have a volume of about 700 #1, into a new tube and then add 4 2 / z l of 100% TCA, which is a 6% final solution. Mix and incubate on ice for 15 min. 21 A. Wach, A. Brachat, C. Alberti-Segui,C. Rebischung,and P. Philippsen,Yeast 13, 1065 (1997). 22M. S. Longtine,A. McKenzieIII, D. J. Demarini,N. G. Shah, A. Wach, A. Brachat, E Philippsen, and J. R. Pringle, Yeast 14, 953 (1998). 23j. Bahler, J. Q. Wu, M. S. Longtine, N. G. Shah, A. McKenzie III, A. B. Steever, A. Wach, E Philippsen,and J. R. Pringle, Yeast 14, 943 (1998). 24O. Puig, B. Rutz, B. G. Luukkonen,S. Kandels-Lewis,E. Bragado-Nilsson,and B. Seraphin,Yeast 14, 1139 (1998). 25M. D. Krawchukand W. E Wahls, Yeast 15, 1419 (1999). 26G. Rigaut, A. Shevchenko,B. Rutz, M. Wilm,M. Mann,and B. Seraphin,Nat. Biotechnol. 17, 1030 (1999). 27A. De Antoniand D. Gallwitz,Gene 246, 179 (2000). 28A. Brachat, N. Liebundguth,C. Rebischung,S. Lemire, E Scharer, D. Hoepfner, V. Demchyshyn, I. Howard, A. Dusterhoft, D. Mostl et al., Yeast 16, 241 (2000). z9 S. Honey,B. L. Schneider,D. M. Schieltz,J. R. Yates, and B. Futcher,Nucleic Acids Res. 29, E24 (2OOl). 30A. L. Goldsteinand J. H. McCusker, Yeast 15, 1541 (1999). 31 M. P. Yaffe and G. Schatz, Proc. Natl. Acad. Sci. U.S.A. 81, 4819 (1984).

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5. Spin down for l0 min at 4 ° at 13,000g and remove the supernatant. Wash the pellet with 1 ml ice-cold acetone. This helps remove acids and salts. 6. Spin down for 10 min at 4 ° at 13,000g and air dry the pellet. 7. Resuspend pellet in SDS-PAGE protein sample buffer. The TCA pellet can be difficult to resuspend, and it may be necessary to work the pellet into solution with a pipette tip. If the protein sample buffer turns yellow, add 2 M Tris-base that has not been adjusted for pH, 1 /zl at a time, until it turns blue again. Be sure to add an equal amount of Tris-base to each sample as the extra salt can cause the samples to run differently on the SDS-PAGE gel. Coimmunoprecipitation Coimmunoprecipitation (co-IP) is a simple and effective method for detecting or confirming a physical interaction between two proteins. 3'18'32 There are a wide variety of protocols for co-IPs, and here we present one sample protocol. The amount of extract or column and/or gradient fraction to be used and the amount of antibody necessary for efficient and effective immunoprecipitation can vary widely and has to be determined empirically for each experiment. A starting point is to use 1 #1 for polyclonal antibodies (serum solutions usually have 0.5 mg/ml or less of the specific antibodies of an antigen or 50 #1 of tissue culture supernatant (supernatants usually have 0.05 mg/ml or less of the specific antibody of an antigen) or 0.5 #1 of ascites fluid (ascites fluid usually has 0.5 mg/ml or less of the specific antibody of an antigen with 2 mg of total protein per reaction (200 #1 from a 10-mg/ml extract3). 1. Add the appropriate amount of antibody to the extract or fraction and incubate on ice at 4 ° for 1 hr to allow binding of antibody to epitopes. Spin extract + antibody for 30 sec at 4 ° at 10,000g to remove any aggregates. 2. Prepare protein A (Sigma) or protein G-Sepharose beads as recommended by the manufacturer in a 1 : l slurry in immunoprecipitation buffer. 3. Add protein A-Sepharose beads (1/10 of the volume of the extract) and tumble on a rotator for 1 hr at 4 ° (i.e., a Barnstead/Thermolyne Labquake Shaker/ Rotisserie, Dubuque, IA). 4. Pellet beads for 30 sec at 4 ° at 10,000g. Save a fraction of the supernatant to determine if the immunoprecipitation depleted the protein of interest from the extract. Aspirate the supernatant with a bent 20-gauge needle to ensure a slow flow and preserve the pellet (we bend the needle twice to make an "N" shape). Wash five times with at least 1 volume of lysis buffer + protease inhibitors. 5. After the last wash, cut off the end of a pipette tip to make the opening larger and pipette the beads into a new Eppendorf tube. This will reduce the amount of nonspecific proteins, which are stuck to the sides of the original tube. 321~A. Kolodziej and R. A. Young, in "Guide to Yeast Genetics and Molecular Biology" (C. Guthrie and G. R. Fink, ed.). AcademicPress, San Diego, 1991.

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6. Resuspend the bead pellet in protein sample buffer plus 2-mercaptoethanol and boil for 5 min (or heat to 65 ° for 20 min). Run an SDS-PAGE gel including some or all of these controls: lysates of untagged strains, antibody itself, bead-only control, antibody-only control, post-IP superuatant, and low- and high-speed supernatants of extracts. The samples can also be frozen at - 20 ° prior to Western blotting. Run only half of each sample and save the other half in case there is a gel disaster. Conclusion We have described in detail how to estimate the molecular weight and shape of soluble proteins from yeast. These estimates, when coupled with epitope tagging and coimmunoprecipitation, give insight into the physical properties and molecular makeup of protein complexes. These estimates can also be very informative when pure recombinant protein is compared with the native protein. The strength of these methods lies in the fact that knowledge of only a single biochemical activity, even as simple as a Western blot, is sufficient to gain some insight into molecular composition and structure. These methods may also be applied to biological complexes with mixed classes of components such as protein-RNA complexes. Finally, the results of size and shape measurement are quantitative and thus can be compared directly with previously determined experimental values. Such comparisons can be very powerful and can provide insight into molecular structure and function. Acknowledgments We thank an astute reviewer and T. Wolkow for very helpful suggestions and critically reading the manuscript. We also thank M. Longtine and T. Mitchison for helpful conversations, J. Huang and L. Lee for outlines of the coimmunoprecipitation and glass bead lysis protocols, and Y. L. Juang for the outline of the denaturing lysis protocol.

[91 P u r i f i c a t i o n

of Glutathione S-Transferase Proteins from Yeast

Fusion

By AVITALA. RODAL, MARA DUNCAN, and DAVID DRUBIN Introduction Purification of affinity-tagged recombinant proteins from prokaryotic hosts is an efficient method to obtain milligram quantities of protein for binding and activity studies. Unfortunately, eukaryotic proteins often are not expressed or are not soluble in prokaryotic hosts, making it necessary to use a eukaryotic expression system such as baculovirus, which can be time-consuming and require access to tissue culture facilities. Yeast provides an opportunity to express large amounts of

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