- Email: [email protected]
[30] Mapping interaction sites between rhodopsin and arrestin by phage display and synthetic peptides
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proteins are run on a gel, where even modest (<20%) amounts of proteolytic products clearly indicate that the protein does not fold properly and/or denatures. In some cases doing the "run-off" and subsequent binding assay at 22 ° helps, but we were never able to express certain bovine visual arrestin mutants and one of the salamander arrestins in a functional form by in vitro translation (although some of these "troublesome" proteins express very well in E. coli). Nonspecific "binding" actually consists of at least two major components: aggregated arrestin that elutes on Sepharose 2B together with rhodopsin-containing membranes and arrestin bound to membranes themselves. With the majority of wild-type and mutant arrestins, most of the nonspecific binding is due to aggregation (i.e., roughly the same levels of nonspecific "binding" is observed in the presence or absence of liposomes), and overall nonspecific binding is less than 1-2% of total arrestin present in the assay. However, some mutants and splice variants, especially those with COOH-terminal truncations, demonstrate substantial binding to liposomes. Because such behavior usually correlates with a high propensity to aggregate, we believe that the phospholipid membranes simply facilitate the denaturation of these proteins. Decreasing the incubation temperature to 20-25 ° and increasing the ionic strength of the column buffer (e.g., using 100 mM NaC1, 10 mM Tris-HCl, pH 7.5, instead of 20 mM Tris-HC1) usually helps, but in some cases the proteins themselves are so unstable and prone to aggregation that it cannot be helped.
[30] M a p p i n g I n t e r a c t i o n S i t e s b e t w e e n R h o d o p s i n a n d Arrestin by Phage Display and Synthetic Peptides
By W. CLAY SMITH and PAUL A. HARGRAVE Introduction Protein-protein interactions form the underlying basis for activation of the components of the phototransduction cascade. Photoactivated rhodopsin interacts with transducin, leading to transducin activation, which then binds and activates cGMP phosphodiesterase. The inactivation process also hinges on protein-protein interactions, starting with the binding of rhodopsin kinase to photoactivated rhodopsin (R*), which then phosphorylates R*. Phosphorylated, activated rhodopsin ( R ' P ) is bound by arrestin, which sterically blocks access of transducin to R ' P , thereby quenching the rhodopsin component of phototransduction.
METHODS IN ENZYMOLOGY, VOL. 315
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
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Much has been learned about phototransduction using classical and innovative approaches to studying these protein interactions. For example, synthetic peptides matching the sequence of the cytoplasmic loops of rhodopsin were used to demonstrate that transducin interacts with multiple sites on R*, including loop 3-4, loop 5-6, and a small loop in the carboxyterminal tail) In a different approach, antibodies against specific regions of rhodopsin kinase were used to show that the amino-terminal region of rhodopsin kinase binds to R*, but that blocking the amino-terminal terminal part of the protein does not block the kinase domain. 2 Molecular biology has provided a different set of tools for dissecting these interactions. For example, site-directed mutagenesis of rhodopsin has been used to demonstrate that loop 3-4 of rhodopsin contributes to the activation of transducin, 3 and that arrestin likely binds to loop 5-6 with some contribution of loops 1-2 and 3-4. 4,5 In this chapter, we show how two different techniques--phage display and synthetic peptides--can be used to study protein-protein interactions. We attempt to give broad principles regarding the use of these techniques followed by specific application of these tools to the mapping of regions of arrestin that bind to rhodopsin. Use of Phage Display Phage display has rapidly emerged during the 1990s as a powerful method to study protein-protein interactions. George Smith first proposed the idea that the filamentous bacteriophage could be used as an expression host to produce fusions of foreign peptides with any one of several phage coat proteins, and that these fusion proteins should be accessible to other potential ligands. 6 Consequently, phage displaying a potential ligand for a substrate could be enriched over ordinary phage by affinity purification, a process known as panning. The filamentous phage are a class of Escherichia coli phage that contain a single strand of DNA encapsulated by 8-10 coat proteins (Fig. 1). The two proteins most commonly used to display proteins are the major coat i B. KOnig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Natl. Acad. Sci. U,S.A, 86, 6878 (1989), 2 K. Palczewski, J. Buczylko, L. Lebioda, J. W. Crabb, and A. S. Polans, J. Biol. Chem. 268, 6004 (1993). 3 R. R. Franke, B. K6nig, T. P. Sakmar, H. G. Khorana, and K. P. Hofmann, Science 250, 123 (1990). 4 j. G. Krupnick, V. Gurevich, T. Schepers, H. E. Hamm, and J. L. Benovic, J. Biol. Chem. 269, 3226 (1994). 5 D. Raman, S. Osawa, and E. R. Weiss, Invest. Ophthalmol. Vis. Sci. 39, $954 (1998). 6 G. P. Smith, Science 228, 1315 (1985).
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pVI I _ _
--A
~
____
.... pVII & plX
~
~
.... /
[
plIl (5 copies)
pVIII (ca. 2700 copies) FIG. 1. Stereotypical structure of the M13 filamentous phage. The single-stranded DNA is surrounded by approximately 10 different types of coat proteins. Proteins pVlII and pill are most commonly modified for use in phage display.
protein (pVIII), which is present at approximately 2700 copies/particle, and the minor coat protein (pIII), present at 3-5 copies/particle. Protein III is responsible for binding to the mating pilus of E. coli and initiating the infection process. The selection of which coat protein to use for a phage display experiment is usually dictated by the goal of the experiment and by the size of the polypeptide being displayed. Generally, the smaller the protein being displayed, the more tolerant the phage will be of multiple copies. In addition, proteins displayed as fusions with the major coat protein are expressed in many more copies than those fused with plII, thus giving the opportunity to select for protein interactions that might have low affinity but high avidity. If selection of high affinity binders is desired, fusions with plII are preferred, since the maximum number of fusions would be five on each phage. Display of multiple copies of the fusion protein is known as polyvalent display. Selection of high-affinity binders can be further enhanced by the use of the phagemid/helper phage system. In this system, the fusion is made with gene III on a phagemid vector and coexpressed with helper phage that provide an excess of coat proteins (including plII). Under these conditions, phage are produced that on average will display 0-1 copies of the fusion protein (monovalent display). The methods described in the following sections utilize this system since we attempted to bias our results toward isolating regions of arrestin that bind to rhodopsin with high affinity. The strength of phage display lies in its ability to rapidly screen very large (108-109) pools of potential protein ligands against a selected substrate. This substrate can be virtually any protein or nonprotein substance that can be used as an affinity matrix. The ability to use any substance as a target is a decided advantage over two-hybrid systems which require that both the library and the target (also known as the "bait") be not only proteins, but also proteins that can be localized to the nucleus. Readers are directed to
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several recent reviews for further background information on phage display. 7-n Vector Selection
General Considerations The choice of vectors is dictated by the goal of the experiment, such as whether a researcher wants to screen a random peptide library, or target sequences from a specific protein. If random peptides are to be screened, several commercial peptide libraries are available for those interested in identifying short-peptide ligands for their substrate (e.g., 7-met and 12-mer libraries from New England BioLabs, Beverly, MA). For those interested in constructing their own peptide libraries or screening sequences from a specific protein, several companies market phagemid vectors with the genelII D N A included in the vector [e.g., pCANTAB 5E (PharmaciaAmersham, Piscataway, NJ), M13KE (New England BioLabs), and pSKAN-8 (Display Systems Biotech, Vista, CA)]. A number of other companies sell generalized phagemid vectors that can be modified for use in phage display by the addition of the genelII or geneVIII cDNA's (e.g., Life Technologies, Rockville, MD, and Stratagene, La Jolla, CA).
Specific Application For the display of arrestin fragments, we chose to use the pCANTAB 5E vector provided by Pharmacia as part of its recombinant antibody phage kit. This vector is provided linearized at SfiI and NotI sites in the genelII DNA. To accept random blunt fragments of arrestin cDNA, the pCANTAB 5E vector was modified by the introduction of a multiple cloning site including an EcoRV blunt site. Complementary oligonucleotides were synthesized that created SfiI-XhoI-SacI-EcoRV-SacI-NotI sites, annealed, and ligated into pCANTAB 5E to create the pCAN-ECO253 vector used in these studies. Insert Preparation
General Considerations The key to insertion of fragments is to maximize the diversity of the inserts. The first consideration is to maintain proper reading frames. Be7 E. s A. 9 B. 10 D. 11 B.
M. Phizicky and S. Fields, Microbiol. Rev. 59, 94 (1995). Bradbury and A. Cattaneo, Trends Neurosci. 18, 243 (1995). A. Katz, Annu. Rev. Biophys. Biomol. Struct. 26, 27 (1997). R. Wilson and B. B. Finlay, Can. J. MicrobioL 44, 313 (1998). K. Kay, A. V. Kurakin, and R. Hyde-DeRuyscher, Drug Disc, Today 3, 370 (1998).
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cause the insert is being introduced into the middle of either genelII or geneVIII, reading frames must be maintained on both ends. Consequently, use of directional insertion is preferred, because random insertion will give only 1/18 in the proper direction and proper reading frame (1/2 in correct direction, 1/3 in the proper 5' reading frame, and 1/3 in the proper 3' reading frame). Directional cloning works well for preparing libraries of short peptides since synthesis of random oligonucleotides is used to prepare the inserts, and these can be synthesized with the appropriate 5' and 3' adapter sequences. However, for the insertion of randomly generated fragments, such as of a specific cDNA, it is difficult to obtain any type of directional or reading frame orientation. Consequently, it is essential that the primary library be made as diverse as possible so that there is adequate representation of all portions of the gene sequence. Two approaches are generally taken for the preparation of random blunt fragments of a specific cDNA. The first uses DNase I digestion, which in the presence of Mn 2+ cleaves double-stranded D N A nonspecifically to give blunt fragments, a2The concentration of DNase I needs to be adjusted to give the desired amount of cleavage, but a concentration of 0.1 U/txg D N A in 50 mM Tris and 10 mM MnC12 (pH 8.0) for 1-5 rain (25 °) is a good starting point. The second approach uses mechanical cleavage of the cDNA through sonication. This method produces more staggered breaks in the DNA than does DNase I digestion, but in our hands appears to yield a more random representation of the cDNA. Figure 2 shows a comparison of the arrestin cDNA fragmented by sonication (lanes 2-6) and by DNase I (lanes %12). Both appear qualitatively similar; however, the phage library we produced using DNase-treated cDNA showed an inherent bias toward the aminoterminal portion of arrestin, whereas sonication produced a more uniform library (see Library Evaluation section).
Specific Application For the production of the arrestin phage library, 1.3 /zg of pCANECO253 was linearized with 20 U EcoRV (12 hr at 37°) and treated with calf-intestinal alkaline phosphatase (5 U, 2 hr, 37°) to reduce vector selfligation. The background of uncut and unphosphorylated phagemid was confirmed to be acceptably low (i.e., 50 ng of self-ligated vector yielded less than 10 colonies from standard chemical transformation). Arrestin cDNA was prepared for insertion by sonication for 2 min at 45% of maximum output (Microzoon Systems Cell Disruptor with 2-mm probe; Heat Systems Ultrasonics Inc., Farmingdale, NY). This length of sonication was 12S. Anderson, Nucl. Acid Res. 9, 3015 (1981).
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1
2
3
4
5
6
7
8
9
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10 11 12
947564125-
FIG.2. Random fragmentation of bovine arrestin cDNA. Full-length cDNA was fragmented by sonication (lanes 2-6) or by treatment with 0.1 U/tzg DNase I (lanes 7-12) for the indicated length of time (in seconds). Sonication was performed with the sample in an ice-water slurry, and DNase I digestion performed at room temperature. DNA samples subjected to 2 min of sonication were used for constructing the arrestin phage library. Lane 1, lambda DNA standard cut with EcoRI and HindlII. empirically determined to give the m a x i m u m amount of D N A in the 200- to 300-bp range, which targets polypeptides of 67-100 amino acids. The sonicated D N A was ligated into p C A N - E C O 2 5 3 , using 12 reactions containing 50 ng of phagemid, and approximately 80 ng of sonicated D N A . C o m p e t e n t T G 1 E. coli cells were prepared, 13 and each ligation was used to transform 1 ml of c o m p e t e n t cells, plating transformed cells on SOB plates TM with 2% (w/v) glucose and 50 tzg/ml carbenicillin. This m e t h o d yielded 3.8 × 103 independent clones with a background of 5% nonrecombinant.
Library Evaluation General Considerations
Because the ultimate success of panning is dependent on having the potential binder in the library, it is a good idea to have some assessment of the quality of the library. In the case of r a n d o m peptide libraries, 10-20 randomly picked colonies should be sequenced to verify the diversity of the peptides (in this small sample size there should be no identical peptides). 13S. R. Kushner, in "Genetic Engineering" (H. W. Boyer and S. Nicosia, eds.), p. 17. Elsevier/ North Holland, Amsterdam, 1978. 14j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
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For gene fragment libraries, the best evaluation is one that assesses the expression properties of the library. This analysis requires a panel of antibodies directed against various epitopes across the polypeptide in order to demonstrate that each part of the protein is adequately represented in the primary library and that there is not an inherent bias to the library. Unfortunately, few laboratories have available such a diverse panel of antibodies. In the absence of these antibodies, an alternative approach is to ascertain that all portions of the eDNA are well represented. For this analysis, colony lifts of the primary library are prepared and then probed with specific portions of the eDNA in order to determine that each part of the cDNA is present in the library. This type of analysis can also be used to determine if there is an inherent bias to the library.
Specific Application Because antibodies against arrestin are available for only a limited range of epitopes (most targeted toward the carboxy terminus), we evaluated our phage library on the basis of the eDNA fragments that had been inserted into geneIII. Defined portions of arrestin cDNA 100-200 bp in length were prepared by polymerase chain reaction (PCR) that covered the entire coding portion of arrestin. These PCR products were then labeled with [ce32p]dCTP (Oligolabelling Kit, Pharmacia, Piscataway, NJ), and hybridized to duplicate colony lifts containing 1500 colonies/lift in aqueous Denhardt's solution following standard DNA/DNA hybridization procedures.15 The lifts were then exposed to X-ray film, and positive colonies counted for each probe (Table I). Each portion of the arrestin eDNA hybridized to some colonies, indicating that all portions of the arrestin eDNA are present in the phage library. Furthermore, when the number of positive colonies for each probe is normalized to the length of the probe, it is clear that each portion of the arrestin eDNA is represented approximately equally within the library (within one standard deviation from the mean) with the exception of 938-1107, which is slightly underrepresented in the library, and 1090-1212, which is slightly overrepresented in the library. Panning
General Considerations Panning provides a method to selectively enrich for the phage that recognize a potential target substrate. Normally, the enrichment provided 15F. M. Ausubel, R. Brent, R. E. Kingston, D. M. Moore, J. G. Seidman, J. A. Smith, and K. Struhk "Current Protocols in Molecular Biology." Wiley, New York, 1998.
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TABLE I ASSESSMENT OF QUALITY OF ARRESTIN PHAGE LIBRARY BY COLONY HYBRIDIZATIONa Portion of arrestin coding region 1-150 68-249 246-410 409-612 547-762 653-837 810-935 938-1107 1090-1212 x ± S.D.
Fragment length (bp)
No. of colonies
No. of colonies/bp
150 182 165 204 216 185 126 170 123
132 120 131 181 156 147 108 106 153
0.88 0.66 0.79 0.89 0.72 0.79 0.86 0.62 1.24 0.83 ± 0.18
Colony lifts were probed with nine portions of the arrestin cDNA, spanning the entire length of the cDNA, and the number of colonies to which each probe hybridized counted. The final column indicates the number of colonies averaged by the length of each probe.
by a single round of panning is on the order of 102-104, with 103 being quite common. Consequently, multiple cycles must usually be performed in order to be able to identify the specific binders from nonspecific background. In a library that contains 108-9 original clones, this typically means three to five rounds of panning. The basis for panning of any phage library is to have available some mechanism for separating the phage bound to the substrate from those that do not bind. The most common method employed in the literature is to use substrate coated on plastic dishes such as tissue culture flasks or tubes (e.g., Nunc Immunotubes, Fisher Scientific, Pittsburgh, PA). Under these circumstances, unbound phage can be simply aspirated from the surface, and the substrate rinsed. However, if it is important that a conformation be preserved in the substrate, then an alternative approach must be used since coating on plastic will denature a protein substrate. One approach taken by several workers is to biotinylate the substrate, thereby allowing for capture of the substrate by streptavidin-coated beadsJ 6A7Commercial biotinylation kits are available (e.g., Pharmacia, Piscataway, NJ) as well as several suppliers of streptavidin coated beads (e.g., Dynal, Lake Success, NY). Importantly, it should be determined empirically if biotinylation influences the binding properties in which the researcher is interested. A final consideration when planning the panning process is the method used to elute the bound phage. Because the phagemid/helper phage system 16 S. P. Parmley and G. P. Smith, Gene 73, 305 (1988). 17 R. E. Hawkins, S. J. Russell, and G. Winter, J. Molec, Biol. 226, 889 (1992).
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produces monovalently displayed pIII fusion polypeptides on average, the remaining pIII proteins are wild type and can directly infect Escherichia coli. Consequently, bacteria can be added to the substrate containing the bound phage and the phage will infect the bacteria. Alternatively, the phage can be eluted from the substrate by mild acid treatment and then added to E. coli for infection. The former method has the advantage of simplicity, and does not run the risk of damaging phage particles by acid treatment (although the filamentous phage are quite resistant to pH extremes of pH 2-12). The direct infection method, however, has two inherent disadvantages. First, direct infection by the addition of bacteria to the bound phage may potentially lose phage that are bound to the substrate with very high affinity, since the affinity of the wild-type pIII is in the nanomolar Kd range. Second, direct infection requires that the phage obtained in each round of panning must be amplified by the bacterial host before being used in the subsequent panning cycle. This reamplification means that there is the potential for the introduction of a bias due to differential growth rates because of the different pIII fusions. Acid elution circumvents the first problem, and can also avoid the second problem if the acid-eluted phage are then used directly in subsequent panning cycles without reamplification. The disadvantage of using the phage directly in subsequent pannings is that if the yield of phage from the previous cycle is low then the total phage recovered after three to four cycles of panning may be vanishingly small. Depending on the yield of phage from the initial panning cycles, it may be necessary to infect and amplify after the first or second panning before proceeding to the next round of panning. Consequently, the optimal approach may use a combination of acid elution and direct infection. Controls
Probably the single most important consideration in successful application of phage display is the use of every conceivable mechanism to reduce background due to binding of the phage to some reaction component other than the target substrate. First, all surfaces on the reaction vessel should be blocked. If the target substrate is applied to plates or tubes, then the unbound surfaces should be blocked after the substrate is applied. We routinely block the microfuge tubes used for panning overnight with 1% (w/v) bovine serum albumin (BSA; Sigma, Fraction V) or 1% (w/v) nonfat powdered milk in phosphate-buffered saline (PBS: 137 mM NaC1, 2.7 mM KCI, 10 mM NaaHPO4, 1.8 mM KH2PO4, pH 7.4). BSA or milk protein should also be included in the panning reaction to help minimize nonspecific binding. However, the phage could bind to these proteins as well; conse-
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quently, the phage library should be preincubated with whichever blocking protein is being used to remove the portion of phage that could bind to these blocking agents. Second, if any material other than the target substrate is present in the panning solution, adequate controls must be used to prevent the phage from binding to this material. For example, in solution-phase panning, the target is often labeled with biotin to allow for capture of the target-phage complex by streptavidin-coated beads. The phage library should be preincubated with both biotin and the streptavidin beads prior to incubation with the target (usually 1 hr at 4° is sufficient). Finally, phage tend to aggregate. Therefore, all phage-containing solutions should be filtered (0.45-tzm filter) to remove these aggregates; single phage will easily pass through this pore size.
Specific Application We wanted to pan our arrestin phage library against R*P with the goal being to isolate the predominant region of arrestin that bound to R*P. Because the conformation of rhodopsin may be an important constraint for identifying the particular binding region we needed rhodopsin in its undenatured state so that we could prepare photoactivated and phosphorylated rhodopsin against which to pan our library. Consequently, we could not use rhodopsin-coated tubes. However, because rhodopsin comprises 95% of the protein in rod disk membranes, 18 we were able to use disk membrane preparations and could sediment these membranes in order to separate the arrestin-phage that bound to R*P from those that did not. Because the success of phage display hinges on the panning process, we have provided a detailed protocol that we used so other users may be better able to adapt the process to their study.
Protocol 1. The primary phage library is cultured in 40 ml 2X YT media (10 g/liter yeast extract, 17 g/liter tryptone) containing 50/xg/ml carbenicillin (Sigma, St. Louis, MO) with 2% (w/v) glucose. 2. When the culture reaches OD600n~ of 0.6, M13K07 helper phage (Promega, Madison, WI) are added at a multiplicity of infection of 5 (using the approximation of 5 x 108 cells/unit OD600nm). 3. After allowing the helper phage to infect the cells (1 hr, 37 ° with gentle shaking), the media is exchanged to 2 x YT containing both 18 W. Krebs and H. Kiahn, Exp. Eye Res. 25, 511 (1977).
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5.
6.
7.
8.
9. 10.
11.
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50/xg/ml carbenicillin and 50/~g/ml kanamycin to select for bacteria that contain both the phagemid and helper phage. Phage are allowed to be secreted into the media 12-16 hr (37°). The yield of phage is usually on the order to 101°-1013 particles. The culture supernatant is collected, and phage precipitated by the addition of 1/10 volume 20% (w/v) polyethylene glycol (PEG) 8000/ 2.5 M NaC1 (60 min, 4°). The phage are collected by centrifugation (10,000 rpm, 10 min, 4°) resuspended in 1 ml PBS containing 1% (w/v) BSA (the BSA serves to help block nonspecific binding), and filtered (0.45/xm) to remove any residual bacteria and aggregates of phage. Disk membranes containing 200/xg of phosphorylated rhodopsin 19 are added to the phage, exposed to white light (2 min) to photoactivate the RP, and allowed to incubate with gentle mixing (60 min, 4°). The membrane/phage mixture is centrifuged (16,000g, 10 min, 4°), and the pellet resuspended in 1 ml PBS containing 0.1% (v/v) Tween 20. This washing with detergent is repeated once more, and then twice more with PBS without detergent. Following the final wash, the phage are acid eluted with 400 tzl PBS, pH 4.0 containing 0.5 M NaCI (15 min at room temperature), then neutralized with 400/zl PBS, pH 11.0. The eluted phage are mixed with 800/xl 2× YT with 2% BSA, to which 200/xg RP is added and the panning process repeated. Following each panning cycle, an aliquot of the phage is used to infect TG1 E. coli cells to monitor the number of phage eluted with each cycle. For titration, TG1 cells are grown to OD600nm of 0.3, a serial titration of phage added (usually 10 1-10-5), and the infected cells plated on 2× YT agar plates with 2% glucose and 50/xg/ml carbenicillin. Each panning cycle should give approximately 102- to 103-fold enrichment of phage that bind to the substrate. If the number of infectious phage particles drops below 104, the phage should be reamplified as in step 1 before proceeding to the next cycle of panning. After completing four panning cycles, isolated colonies from the plates are cultured and phagemid DNA prepared following standard minipreparation protocols for the isolation of plasmid DNA (e.g., Wizard Miniprep kits, Promega, Madison, WI). Because the TGI strain of E. coli is not an endonuclease-deficient strain ( E n d A ) ,
~9j. Puig, A. Arendt, F. L. Tomson, G. Abdulaeva, R. Miller, P. A. Hargrave, and J. H. McDowell, FEBS Lett. 362, 185 (1995).
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proteases should be included in the sample preparation buffers in order to improve the quality of D N A for sequencing. We sequenced the phagemid D N A manually using the dideoxy termination method 2° in the presence of [35S]dATP,although automatic sequencing works as well. It is sufficient to sequence across the insertion site from both directions to determine which portion of the arrestin cDNA was cloned into the genelII D N A and to ensure that proper frame orientation is maintained. Any inserts that are not in frame with the genelII open reading frame should be discarded from the data. If only a few colonies are obtained after panning, it is an easy matter to sequence all clones. However, if several thousand colonies are obtained, then only a sample of the colonies can be reasonably sequenced. We obtained 2.4 × 103 colonies from phage panned against R*P in disk membranes and 300 colonies from phage panned against red blood cell membranes. We sequence 200 colonies from the phage panned against R*P and 50 colonies from phage panned against our control membranes. Control Considerations
In our experiment rhodopsin was provided in disk membranes prepared from rod outer segments. This provided the added component of the lipid bilayer in which the rhodopsin was embedded. The best control would have been the same lipid membrane with rhodopsin removed. However, this material is quite difficult to prepare in the quantities required for the panning experiments. Instead, we chose an alternative approach in which membranes were collected from lysed red blood cells. 21 These membranes were used in a parallel panning experiment. From the small number of colonies obtained in this control experiment we were able to conclude that the contribution due to nonspecific binding to membranes was likely to be insignificant. Data Analysis General Considerations
Data analysis is relatively straightforward since it involves simply a compilation and alignment of the sequences obtained that bound to the substrate. For random peptide sequences, the goal is to identify one or a few residues that emerge as a consensus residue in the same location of 20 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 21 D. Hanahan and J. Ekholm, Methods Enzymol. 3L 168 (1974).
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Ao
R*P in Disk Mbrs 25
50
75
100
12S
150
175
200
Z~5
250
275
3z00
325
25
50
75
100
lzs
l~
17s
200
2~s
~50
~75
300
325
RBC
B. t
350
3so
375
404
Mbrs ~TS
FIG. 3. Fragments of arrestin that were displayed on phage that bound (A) to disk membranes containing R*P or (B) to membranes prepared from red blood cells (RBCs). The arrestin polypeptide is indicated as an open bar with amino acid positions numbered above. Each displayed peptide that bound to the substrate is indicated as a single line.
the peptide (usually present in 50-70% of the isolated peptides). For screens of a specific protein, the goal is to isolate the minimal epitope from the overlapping peptides.
Specific Application Figure 3 shows a compilation of the arrestin sequences that bound to R*P in disk m e m b r a n e s (Fig. 3A) and those that b o u n d to red blood cell m e m b r a n e s (Fig. 3B). U n d e r ideal circumstances, a minimal epitope will be revealed that can be used for further studies. In our experiment, the entire molecule of the protein was represented. Perhaps this reflects the fact that multiple portions of arrestin contribute to the binding to rhodopsin.
404
450
PROTEINS THAT INTERACT WITH RHODOPSIN 30
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i
i
i
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i
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i
i
i
i
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i
i
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50
100
150
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200
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250
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300
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FIG. 4. Frequency histogram representing the number of times [per 100 colony-forming units (cfu)] any given amino acid was displayed on a phage that bound to R * P in (A) disk membranes or (B) to m e m b r a n e s from RBCs.
However, if these data are converted to a frequency histogram (Fig. 4), simply calculating the number of times any particular amino acid of arrestin was present in a phage that bound to R'P, it is clear the most prevalent epitope was 90-140, with additional peaks at 160-210 and 240-270. Note that for the primary peak of binding (90-140) there was virtually no binding to red blood cell membranes, indicating that it is unlikely that the phage
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displaying this region of arrestin were binding nonspecifically to the lipid membrane. Data Confirmation General Considerations One critical feature of phage display is that like other library screening techniques, such as yeast two-hybrid or cDNA expression library screening, all potential positives must be tested to eliminate false-positives and establish biological relevance. This can be approached using any number of methods that are appropriate for the particular system, but usually include heterologous expression to produce protein for measuring binding parameters, receptor activation/inactivation assays, or binding competition assays. Specific Application For our arrestin phage display, we chose to express a portion of the principal binding region as a fusion with glutathione S-transferase and demonstrate that this fusion could function as a competitor with arrestin for binding to R*P. We first expressed the region of arrestin spanning amino acids 95-140 as a carboxyl-terminal fusion protein with glutathione S-transferase. The arrestin cDNA corresponding to this region was amplified by PCR, adding 5' EcoRI and 3' Sall restriction sites with synthetic oligonucleotide primers, and cloned into the pGEX-4T-1 vector (Pharmacia) at the EcoRI and SalI sites. This recombinant plasmid was then introduced into BL21 E. coli cells and used to express soluble protein, inducing with 1 mM IPTG (4 hr, 37°). The expressed protein was purified over glutathione-agarose (Pharmacia) following the manufacturer's recommended procedures. We were able to obtain approximately 2 rag/liter of pure protein. We then assayed the ability of this protein to compete for the binding of arrestin to R*P. Arrestin (10/~g) was mixed with R*P (40 ~g) contained in disk membranes in PBS in the presence of increasing amounts (0-1 raM) of 95-140/GST fusion protein, and 1/.~M glyceraldehyde 3-phosphate dehydrogenase in a 150-/~1 reaction volume. (The dehydrogenase serves as an internal control for quantitation.) Following incubation (2 rain, 4°), the reaction mixture was centrifuged (24,000g, 30 min, 4°) to sediment the disk membranes along with any bound arrestin. The amount of arrestin and R*P used was empirically determined to give complete binding of the arrestin in the absence of fusion protein. The reaction supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel
452
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Peptide Concentration ( tM) FI~. 5. Binding inhibition of arrestin to R*P using 95-140/GST fusion protein. Incubations containing 1.4 ~M arrestin, 8/~M R*P in disk membranes, and the indicated amount of fusion protein (open squares) or glutathione S-transferase alone (closed circles) in the presence of 1 t~M glyceraldehyde 3-phosphate dehydrogenase were prepared as described. The amount of arrestin competitively released into the supernatant was measured by SDS-PAGE, stained with Coomassie blue, and quantified by scanning densitometry. The curves represent the means (+ S.D.) from three experiments.
(SDS-PAGE) electrophoresis to determine if any arrestin was displaced by competition into this fraction. The amount of arrestin present in the reaction supernatant was quantified by scanning densitometry (versions of NIH Image are available for free downloading from NIH's website, http://rsb.info.nih.gov/nih-image/), normalizing the samples to the internal glyceraldehyde 3-phosphate control. Figure 5 shows that as the concentration of 95-140/GST is increased, there is an increasing amount of unbound arrestin. Note that GST alone has no influence on the binding of arrestin to R*P. If this competition is projected, the IC50 for the inhibition is projected to be in the low millimolar range. This experiment provides corroboration of the data obtained using phage display.
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Use of Synthetic Peptides Protein-protein interactions form the basis of many key events in biological processes. It is often possible to test synthetic peptides from the sequence of one of the interacting proteins to determine whether they compete in a protein-protein binding reaction, and thus determine the sites of interaction between the proteins. This technique has been successfully applied in several studies of protein interaction of interest in the study of the biochemistry of vision. Some peptides representing sequences displayed on the cytoplasmic surface of rhodopsin compete for the binding of transducin to R*, thus helping delineate binding sites on rhodopsin for transducin. 22 Synthetic peptides corresponding to two regions near the carboxyl terminus of the transducin a subunit compete with transducin for the binding to R*, thus suggesting part of the regions of transduein that interact with R*. 23 The third cytoplasmic loop peptide of rhodopsin competes for the binding of arrestin to R'P, suggesting this loop as a major site of interaction between the two proteins, a4 And the interaction site by which guanylate cyclase becomes activated by its activating protein has been shown to be within the catalytic domain of guanylate cyclase, by peptide competition. 25 Synthetic peptides have also been used to map epitope specificities of libraries of anti-rhodopsin monoclonal antibodies. 26'2v
Specific Application For this research overlapping peptides 20 amino acids in length were synthesized using RAMPS Multiple Peptide Synthesis System (DuPont, Wilmington, DE). Most peptides were synthesized using "Wang" Carboxylate Resin cartridges with 0.1 mmol of first 9-fluoroenylmethoxycarbonyl amino acids on p-alkoxybenzyl alcohol resin (DuPont). Sequential amino acids were coupled according to the RAMPS protocol using HOBt esters prepared in situ from Fmoc amino acids. Cleavage and deprotection from resin was carried out by incubating the resin for 3 hr at room temperature 22 B. K6nig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Natl. Acad. Sci. U.S.A. 86, 6878 (1989). 23 H. E. Hamm, D. Deretic, A. Arendt, P. A. Hargrave, B. Koenig, and K. P. Hofmann, Science 241, 832 (1988). 24 j. G. Krupnick, V. V. Gurevich, T. Schepers, H. E. Hamm, and J. L. Benovic, J. Biol. Chem. 269, 3226 (1994). 25 I. Sokal, F. Haeseleer, A. Arendt, E. T. Adman, P. A. Hargrave, and K. Palczewski, Biochemistry 38, 1387 (1999). 26 R. S. Hodges, R. J. Heaton, J. M. R. Parker, L. Molday, and R. S. Molday, J. Biol. Chem. 263, 11768 (1988). 27 G. Adamus, Z. S. Zam, A. Arendt, K. Palczewski, J. H. McDowell, and P. A. Hargrave, Vis. Res. 31, 17 (1991).
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PROTEINS THAT INTERACT WITH RHODOPSIN
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F~o. 6. Binding competition of arrestin to phosphorylated photoactivated rhodopsin using synthetic arrestin peptides. Photoactivated disk membranes containing 8/xM R*P were incubated with 1.4/zM arrestin in the presence of 700/~M syntheticpeptides. Followingcentrifugation, inhibition of arrestin bindingwas measured by analyzingthe amount of arrestin remaining in the supernatant using SDS-PAGE. Arrestin was quantified by scanning densitometry, normalizing arrestin to an internal standard (glyceraldehyde 3-phosphate dehydrogenase). The means (-+ S.D.) from three experiments are shown. in 90: 5 : 5 (v/v) trifluoroacetic acid (TFA) : H 2 0 : ethanedithiol. For purification, peptides were precipitated from solution using cold diethyl ether, lyophilized, and further purified over Bio-Gel P-2 (Bio-Rad, Hercules, CA) in 5% (v/v) acetic acid. Quality was assessed by amino acid analyses and analytical HPLC. The peptides synthesized corresponded to arrestin sequence 81-100, 91-110, 101-120, 111-130, 120-140, 131-150, 141-160, and 151-170. The peptides were tested in a binding competition assay, using the same methodology as delineated earlier for testing the effect of 95-140/GST fusion on arrestin binding to R*P except that increasing concentrations of peptides (0-700 /zM) were used instead of the fusion protein. Figure 6 shows the amount of arrestin binding that was inhibited by each peptide. These results show some effect on binding by peptides 101-120, 111-130,
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and 121-140, with the largest effect by 111-130. These data clearly implicate the region 101-130 as containing a binding domain, which corresponds nicely with the phage display data. Because 111-130 has limited solubility in aqueous buffers, this peptide was resynthesized to include Lys-109 and Lys-ll0, as well as to substitute Cys-128 with serine (i.e., 109-130/Cys128 Ser). Using this peptide, we were able to completely block the binding of arrestin to R*P with an ICs0 of 1.1 mM. The relatively high IC50 likely indicates that arrestin has multiple points of contact with R ' P , which is consistent with the observations from other laboratories. 28-3° Note that a peptide containing the same amino acid composition, but ordered randomly (i.e., KDYLVPLSSNAPLYLGTFPFTK) was not an effective competitor. In conclusion, the use of synthetic peptides identifies a binding region in arrestin for R*P that is composed of, at least in part, amino acids 111-130. These data are consistent with those obtained using phage display. Consequently, the synthetic peptides are useful as additional corroboration of the phage display study, but also provide a more narrow definition of the binding region. Acknowledgment This research was supported by a Career D e v e l o p m e n t Award from the Research to Prevent Blindness (RPB) Foundation to W.C.S., a Senior Scientific Investigator award from R P B to P A H , grants from the National Eye Institute (EY06225, EY06226, EY0857l). and an unrestricted grant to the D e p a r t m e n t of O p h t h a l m o l o g y from RPB. _,s V. V. Gurevich and J. L. Benovic, J. BioL Chem. 268, 11628 (1993). 29 V. V. Gurevich and J. L. Benovic, J. Biol. Chem., 270, 6010 (1995). 3~ K. Palczewski, J. Buczylko, N. R. Imami, J. H. McDowell, and P. A. Hargrave, J. Biol. Chem. 266, 15334 (1991).
[31] C h a r a c t e r i z a t i o n o f R a n B P 2 - A s s o c i a t e d Components in Neuroretina
Molecular
By PAULO A. FERREIRA Introduction In search of proteins that mediate the biogenesis of vertebrate Gprotein-coupled light receptors, opsins, we have cloned several products I I p. Ferreira, J. H o m , and W. Pak, J. BioL Chem. 270, 23179 (1995).
METHODS IN ENZYMOLOGY,VOL. 315
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proteins are run on a gel, where even modest (<20%) amounts of proteolytic products clearly indicate that the protein does not fold properly and/or denatures. In some cases doing the "run-off" and subsequent binding assay at 22 ° helps, but we were never able to express certain bovine visual arrestin mutants and one of the salamander arrestins in a functional form by in vitro translation (although some of these "troublesome" proteins express very well in E. coli). Nonspecific "binding" actually consists of at least two major components: aggregated arrestin that elutes on Sepharose 2B together with rhodopsin-containing membranes and arrestin bound to membranes themselves. With the majority of wild-type and mutant arrestins, most of the nonspecific binding is due to aggregation (i.e., roughly the same levels of nonspecific "binding" is observed in the presence or absence of liposomes), and overall nonspecific binding is less than 1-2% of total arrestin present in the assay. However, some mutants and splice variants, especially those with COOH-terminal truncations, demonstrate substantial binding to liposomes. Because such behavior usually correlates with a high propensity to aggregate, we believe that the phospholipid membranes simply facilitate the denaturation of these proteins. Decreasing the incubation temperature to 20-25 ° and increasing the ionic strength of the column buffer (e.g., using 100 mM NaC1, 10 mM Tris-HCl, pH 7.5, instead of 20 mM Tris-HC1) usually helps, but in some cases the proteins themselves are so unstable and prone to aggregation that it cannot be helped.
[30] M a p p i n g I n t e r a c t i o n S i t e s b e t w e e n R h o d o p s i n a n d Arrestin by Phage Display and Synthetic Peptides
By W. CLAY SMITH and PAUL A. HARGRAVE Introduction Protein-protein interactions form the underlying basis for activation of the components of the phototransduction cascade. Photoactivated rhodopsin interacts with transducin, leading to transducin activation, which then binds and activates cGMP phosphodiesterase. The inactivation process also hinges on protein-protein interactions, starting with the binding of rhodopsin kinase to photoactivated rhodopsin (R*), which then phosphorylates R*. Phosphorylated, activated rhodopsin ( R ' P ) is bound by arrestin, which sterically blocks access of transducin to R ' P , thereby quenching the rhodopsin component of phototransduction.
METHODS IN ENZYMOLOGY, VOL. 315
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Much has been learned about phototransduction using classical and innovative approaches to studying these protein interactions. For example, synthetic peptides matching the sequence of the cytoplasmic loops of rhodopsin were used to demonstrate that transducin interacts with multiple sites on R*, including loop 3-4, loop 5-6, and a small loop in the carboxyterminal tail) In a different approach, antibodies against specific regions of rhodopsin kinase were used to show that the amino-terminal region of rhodopsin kinase binds to R*, but that blocking the amino-terminal terminal part of the protein does not block the kinase domain. 2 Molecular biology has provided a different set of tools for dissecting these interactions. For example, site-directed mutagenesis of rhodopsin has been used to demonstrate that loop 3-4 of rhodopsin contributes to the activation of transducin, 3 and that arrestin likely binds to loop 5-6 with some contribution of loops 1-2 and 3-4. 4,5 In this chapter, we show how two different techniques--phage display and synthetic peptides--can be used to study protein-protein interactions. We attempt to give broad principles regarding the use of these techniques followed by specific application of these tools to the mapping of regions of arrestin that bind to rhodopsin. Use of Phage Display Phage display has rapidly emerged during the 1990s as a powerful method to study protein-protein interactions. George Smith first proposed the idea that the filamentous bacteriophage could be used as an expression host to produce fusions of foreign peptides with any one of several phage coat proteins, and that these fusion proteins should be accessible to other potential ligands. 6 Consequently, phage displaying a potential ligand for a substrate could be enriched over ordinary phage by affinity purification, a process known as panning. The filamentous phage are a class of Escherichia coli phage that contain a single strand of DNA encapsulated by 8-10 coat proteins (Fig. 1). The two proteins most commonly used to display proteins are the major coat i B. KOnig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Natl. Acad. Sci. U,S.A, 86, 6878 (1989), 2 K. Palczewski, J. Buczylko, L. Lebioda, J. W. Crabb, and A. S. Polans, J. Biol. Chem. 268, 6004 (1993). 3 R. R. Franke, B. K6nig, T. P. Sakmar, H. G. Khorana, and K. P. Hofmann, Science 250, 123 (1990). 4 j. G. Krupnick, V. Gurevich, T. Schepers, H. E. Hamm, and J. L. Benovic, J. Biol. Chem. 269, 3226 (1994). 5 D. Raman, S. Osawa, and E. R. Weiss, Invest. Ophthalmol. Vis. Sci. 39, $954 (1998). 6 G. P. Smith, Science 228, 1315 (1985).
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pVI I _ _
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~
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pVIII (ca. 2700 copies) FIG. 1. Stereotypical structure of the M13 filamentous phage. The single-stranded DNA is surrounded by approximately 10 different types of coat proteins. Proteins pVlII and pill are most commonly modified for use in phage display.
protein (pVIII), which is present at approximately 2700 copies/particle, and the minor coat protein (pIII), present at 3-5 copies/particle. Protein III is responsible for binding to the mating pilus of E. coli and initiating the infection process. The selection of which coat protein to use for a phage display experiment is usually dictated by the goal of the experiment and by the size of the polypeptide being displayed. Generally, the smaller the protein being displayed, the more tolerant the phage will be of multiple copies. In addition, proteins displayed as fusions with the major coat protein are expressed in many more copies than those fused with plII, thus giving the opportunity to select for protein interactions that might have low affinity but high avidity. If selection of high affinity binders is desired, fusions with plII are preferred, since the maximum number of fusions would be five on each phage. Display of multiple copies of the fusion protein is known as polyvalent display. Selection of high-affinity binders can be further enhanced by the use of the phagemid/helper phage system. In this system, the fusion is made with gene III on a phagemid vector and coexpressed with helper phage that provide an excess of coat proteins (including plII). Under these conditions, phage are produced that on average will display 0-1 copies of the fusion protein (monovalent display). The methods described in the following sections utilize this system since we attempted to bias our results toward isolating regions of arrestin that bind to rhodopsin with high affinity. The strength of phage display lies in its ability to rapidly screen very large (108-109) pools of potential protein ligands against a selected substrate. This substrate can be virtually any protein or nonprotein substance that can be used as an affinity matrix. The ability to use any substance as a target is a decided advantage over two-hybrid systems which require that both the library and the target (also known as the "bait") be not only proteins, but also proteins that can be localized to the nucleus. Readers are directed to
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several recent reviews for further background information on phage display. 7-n Vector Selection
General Considerations The choice of vectors is dictated by the goal of the experiment, such as whether a researcher wants to screen a random peptide library, or target sequences from a specific protein. If random peptides are to be screened, several commercial peptide libraries are available for those interested in identifying short-peptide ligands for their substrate (e.g., 7-met and 12-mer libraries from New England BioLabs, Beverly, MA). For those interested in constructing their own peptide libraries or screening sequences from a specific protein, several companies market phagemid vectors with the genelII D N A included in the vector [e.g., pCANTAB 5E (PharmaciaAmersham, Piscataway, NJ), M13KE (New England BioLabs), and pSKAN-8 (Display Systems Biotech, Vista, CA)]. A number of other companies sell generalized phagemid vectors that can be modified for use in phage display by the addition of the genelII or geneVIII cDNA's (e.g., Life Technologies, Rockville, MD, and Stratagene, La Jolla, CA).
Specific Application For the display of arrestin fragments, we chose to use the pCANTAB 5E vector provided by Pharmacia as part of its recombinant antibody phage kit. This vector is provided linearized at SfiI and NotI sites in the genelII DNA. To accept random blunt fragments of arrestin cDNA, the pCANTAB 5E vector was modified by the introduction of a multiple cloning site including an EcoRV blunt site. Complementary oligonucleotides were synthesized that created SfiI-XhoI-SacI-EcoRV-SacI-NotI sites, annealed, and ligated into pCANTAB 5E to create the pCAN-ECO253 vector used in these studies. Insert Preparation
General Considerations The key to insertion of fragments is to maximize the diversity of the inserts. The first consideration is to maintain proper reading frames. Be7 E. s A. 9 B. 10 D. 11 B.
M. Phizicky and S. Fields, Microbiol. Rev. 59, 94 (1995). Bradbury and A. Cattaneo, Trends Neurosci. 18, 243 (1995). A. Katz, Annu. Rev. Biophys. Biomol. Struct. 26, 27 (1997). R. Wilson and B. B. Finlay, Can. J. MicrobioL 44, 313 (1998). K. Kay, A. V. Kurakin, and R. Hyde-DeRuyscher, Drug Disc, Today 3, 370 (1998).
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cause the insert is being introduced into the middle of either genelII or geneVIII, reading frames must be maintained on both ends. Consequently, use of directional insertion is preferred, because random insertion will give only 1/18 in the proper direction and proper reading frame (1/2 in correct direction, 1/3 in the proper 5' reading frame, and 1/3 in the proper 3' reading frame). Directional cloning works well for preparing libraries of short peptides since synthesis of random oligonucleotides is used to prepare the inserts, and these can be synthesized with the appropriate 5' and 3' adapter sequences. However, for the insertion of randomly generated fragments, such as of a specific cDNA, it is difficult to obtain any type of directional or reading frame orientation. Consequently, it is essential that the primary library be made as diverse as possible so that there is adequate representation of all portions of the gene sequence. Two approaches are generally taken for the preparation of random blunt fragments of a specific cDNA. The first uses DNase I digestion, which in the presence of Mn 2+ cleaves double-stranded D N A nonspecifically to give blunt fragments, a2The concentration of DNase I needs to be adjusted to give the desired amount of cleavage, but a concentration of 0.1 U/txg D N A in 50 mM Tris and 10 mM MnC12 (pH 8.0) for 1-5 rain (25 °) is a good starting point. The second approach uses mechanical cleavage of the cDNA through sonication. This method produces more staggered breaks in the DNA than does DNase I digestion, but in our hands appears to yield a more random representation of the cDNA. Figure 2 shows a comparison of the arrestin cDNA fragmented by sonication (lanes 2-6) and by DNase I (lanes %12). Both appear qualitatively similar; however, the phage library we produced using DNase-treated cDNA showed an inherent bias toward the aminoterminal portion of arrestin, whereas sonication produced a more uniform library (see Library Evaluation section).
Specific Application For the production of the arrestin phage library, 1.3 /zg of pCANECO253 was linearized with 20 U EcoRV (12 hr at 37°) and treated with calf-intestinal alkaline phosphatase (5 U, 2 hr, 37°) to reduce vector selfligation. The background of uncut and unphosphorylated phagemid was confirmed to be acceptably low (i.e., 50 ng of self-ligated vector yielded less than 10 colonies from standard chemical transformation). Arrestin cDNA was prepared for insertion by sonication for 2 min at 45% of maximum output (Microzoon Systems Cell Disruptor with 2-mm probe; Heat Systems Ultrasonics Inc., Farmingdale, NY). This length of sonication was 12S. Anderson, Nucl. Acid Res. 9, 3015 (1981).
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2
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10 11 12
947564125-
FIG.2. Random fragmentation of bovine arrestin cDNA. Full-length cDNA was fragmented by sonication (lanes 2-6) or by treatment with 0.1 U/tzg DNase I (lanes 7-12) for the indicated length of time (in seconds). Sonication was performed with the sample in an ice-water slurry, and DNase I digestion performed at room temperature. DNA samples subjected to 2 min of sonication were used for constructing the arrestin phage library. Lane 1, lambda DNA standard cut with EcoRI and HindlII. empirically determined to give the m a x i m u m amount of D N A in the 200- to 300-bp range, which targets polypeptides of 67-100 amino acids. The sonicated D N A was ligated into p C A N - E C O 2 5 3 , using 12 reactions containing 50 ng of phagemid, and approximately 80 ng of sonicated D N A . C o m p e t e n t T G 1 E. coli cells were prepared, 13 and each ligation was used to transform 1 ml of c o m p e t e n t cells, plating transformed cells on SOB plates TM with 2% (w/v) glucose and 50 tzg/ml carbenicillin. This m e t h o d yielded 3.8 × 103 independent clones with a background of 5% nonrecombinant.
Library Evaluation General Considerations
Because the ultimate success of panning is dependent on having the potential binder in the library, it is a good idea to have some assessment of the quality of the library. In the case of r a n d o m peptide libraries, 10-20 randomly picked colonies should be sequenced to verify the diversity of the peptides (in this small sample size there should be no identical peptides). 13S. R. Kushner, in "Genetic Engineering" (H. W. Boyer and S. Nicosia, eds.), p. 17. Elsevier/ North Holland, Amsterdam, 1978. 14j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
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For gene fragment libraries, the best evaluation is one that assesses the expression properties of the library. This analysis requires a panel of antibodies directed against various epitopes across the polypeptide in order to demonstrate that each part of the protein is adequately represented in the primary library and that there is not an inherent bias to the library. Unfortunately, few laboratories have available such a diverse panel of antibodies. In the absence of these antibodies, an alternative approach is to ascertain that all portions of the eDNA are well represented. For this analysis, colony lifts of the primary library are prepared and then probed with specific portions of the eDNA in order to determine that each part of the cDNA is present in the library. This type of analysis can also be used to determine if there is an inherent bias to the library.
Specific Application Because antibodies against arrestin are available for only a limited range of epitopes (most targeted toward the carboxy terminus), we evaluated our phage library on the basis of the eDNA fragments that had been inserted into geneIII. Defined portions of arrestin cDNA 100-200 bp in length were prepared by polymerase chain reaction (PCR) that covered the entire coding portion of arrestin. These PCR products were then labeled with [ce32p]dCTP (Oligolabelling Kit, Pharmacia, Piscataway, NJ), and hybridized to duplicate colony lifts containing 1500 colonies/lift in aqueous Denhardt's solution following standard DNA/DNA hybridization procedures.15 The lifts were then exposed to X-ray film, and positive colonies counted for each probe (Table I). Each portion of the arrestin eDNA hybridized to some colonies, indicating that all portions of the arrestin eDNA are present in the phage library. Furthermore, when the number of positive colonies for each probe is normalized to the length of the probe, it is clear that each portion of the arrestin eDNA is represented approximately equally within the library (within one standard deviation from the mean) with the exception of 938-1107, which is slightly underrepresented in the library, and 1090-1212, which is slightly overrepresented in the library. Panning
General Considerations Panning provides a method to selectively enrich for the phage that recognize a potential target substrate. Normally, the enrichment provided 15F. M. Ausubel, R. Brent, R. E. Kingston, D. M. Moore, J. G. Seidman, J. A. Smith, and K. Struhk "Current Protocols in Molecular Biology." Wiley, New York, 1998.
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TABLE I ASSESSMENT OF QUALITY OF ARRESTIN PHAGE LIBRARY BY COLONY HYBRIDIZATIONa Portion of arrestin coding region 1-150 68-249 246-410 409-612 547-762 653-837 810-935 938-1107 1090-1212 x ± S.D.
Fragment length (bp)
No. of colonies
No. of colonies/bp
150 182 165 204 216 185 126 170 123
132 120 131 181 156 147 108 106 153
0.88 0.66 0.79 0.89 0.72 0.79 0.86 0.62 1.24 0.83 ± 0.18
Colony lifts were probed with nine portions of the arrestin cDNA, spanning the entire length of the cDNA, and the number of colonies to which each probe hybridized counted. The final column indicates the number of colonies averaged by the length of each probe.
by a single round of panning is on the order of 102-104, with 103 being quite common. Consequently, multiple cycles must usually be performed in order to be able to identify the specific binders from nonspecific background. In a library that contains 108-9 original clones, this typically means three to five rounds of panning. The basis for panning of any phage library is to have available some mechanism for separating the phage bound to the substrate from those that do not bind. The most common method employed in the literature is to use substrate coated on plastic dishes such as tissue culture flasks or tubes (e.g., Nunc Immunotubes, Fisher Scientific, Pittsburgh, PA). Under these circumstances, unbound phage can be simply aspirated from the surface, and the substrate rinsed. However, if it is important that a conformation be preserved in the substrate, then an alternative approach must be used since coating on plastic will denature a protein substrate. One approach taken by several workers is to biotinylate the substrate, thereby allowing for capture of the substrate by streptavidin-coated beadsJ 6A7Commercial biotinylation kits are available (e.g., Pharmacia, Piscataway, NJ) as well as several suppliers of streptavidin coated beads (e.g., Dynal, Lake Success, NY). Importantly, it should be determined empirically if biotinylation influences the binding properties in which the researcher is interested. A final consideration when planning the panning process is the method used to elute the bound phage. Because the phagemid/helper phage system 16 S. P. Parmley and G. P. Smith, Gene 73, 305 (1988). 17 R. E. Hawkins, S. J. Russell, and G. Winter, J. Molec, Biol. 226, 889 (1992).
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produces monovalently displayed pIII fusion polypeptides on average, the remaining pIII proteins are wild type and can directly infect Escherichia coli. Consequently, bacteria can be added to the substrate containing the bound phage and the phage will infect the bacteria. Alternatively, the phage can be eluted from the substrate by mild acid treatment and then added to E. coli for infection. The former method has the advantage of simplicity, and does not run the risk of damaging phage particles by acid treatment (although the filamentous phage are quite resistant to pH extremes of pH 2-12). The direct infection method, however, has two inherent disadvantages. First, direct infection by the addition of bacteria to the bound phage may potentially lose phage that are bound to the substrate with very high affinity, since the affinity of the wild-type pIII is in the nanomolar Kd range. Second, direct infection requires that the phage obtained in each round of panning must be amplified by the bacterial host before being used in the subsequent panning cycle. This reamplification means that there is the potential for the introduction of a bias due to differential growth rates because of the different pIII fusions. Acid elution circumvents the first problem, and can also avoid the second problem if the acid-eluted phage are then used directly in subsequent panning cycles without reamplification. The disadvantage of using the phage directly in subsequent pannings is that if the yield of phage from the previous cycle is low then the total phage recovered after three to four cycles of panning may be vanishingly small. Depending on the yield of phage from the initial panning cycles, it may be necessary to infect and amplify after the first or second panning before proceeding to the next round of panning. Consequently, the optimal approach may use a combination of acid elution and direct infection. Controls
Probably the single most important consideration in successful application of phage display is the use of every conceivable mechanism to reduce background due to binding of the phage to some reaction component other than the target substrate. First, all surfaces on the reaction vessel should be blocked. If the target substrate is applied to plates or tubes, then the unbound surfaces should be blocked after the substrate is applied. We routinely block the microfuge tubes used for panning overnight with 1% (w/v) bovine serum albumin (BSA; Sigma, Fraction V) or 1% (w/v) nonfat powdered milk in phosphate-buffered saline (PBS: 137 mM NaC1, 2.7 mM KCI, 10 mM NaaHPO4, 1.8 mM KH2PO4, pH 7.4). BSA or milk protein should also be included in the panning reaction to help minimize nonspecific binding. However, the phage could bind to these proteins as well; conse-
446
PROTEINS THAT INTERACT WITH RHODOPSIN
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quently, the phage library should be preincubated with whichever blocking protein is being used to remove the portion of phage that could bind to these blocking agents. Second, if any material other than the target substrate is present in the panning solution, adequate controls must be used to prevent the phage from binding to this material. For example, in solution-phase panning, the target is often labeled with biotin to allow for capture of the target-phage complex by streptavidin-coated beads. The phage library should be preincubated with both biotin and the streptavidin beads prior to incubation with the target (usually 1 hr at 4° is sufficient). Finally, phage tend to aggregate. Therefore, all phage-containing solutions should be filtered (0.45-tzm filter) to remove these aggregates; single phage will easily pass through this pore size.
Specific Application We wanted to pan our arrestin phage library against R*P with the goal being to isolate the predominant region of arrestin that bound to R*P. Because the conformation of rhodopsin may be an important constraint for identifying the particular binding region we needed rhodopsin in its undenatured state so that we could prepare photoactivated and phosphorylated rhodopsin against which to pan our library. Consequently, we could not use rhodopsin-coated tubes. However, because rhodopsin comprises 95% of the protein in rod disk membranes, 18 we were able to use disk membrane preparations and could sediment these membranes in order to separate the arrestin-phage that bound to R*P from those that did not. Because the success of phage display hinges on the panning process, we have provided a detailed protocol that we used so other users may be better able to adapt the process to their study.
Protocol 1. The primary phage library is cultured in 40 ml 2X YT media (10 g/liter yeast extract, 17 g/liter tryptone) containing 50/xg/ml carbenicillin (Sigma, St. Louis, MO) with 2% (w/v) glucose. 2. When the culture reaches OD600n~ of 0.6, M13K07 helper phage (Promega, Madison, WI) are added at a multiplicity of infection of 5 (using the approximation of 5 x 108 cells/unit OD600nm). 3. After allowing the helper phage to infect the cells (1 hr, 37 ° with gentle shaking), the media is exchanged to 2 x YT containing both 18 W. Krebs and H. Kiahn, Exp. Eye Res. 25, 511 (1977).
[30]
MAPPING INTERACTION SITES
4.
5.
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7.
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11.
447
50/xg/ml carbenicillin and 50/~g/ml kanamycin to select for bacteria that contain both the phagemid and helper phage. Phage are allowed to be secreted into the media 12-16 hr (37°). The yield of phage is usually on the order to 101°-1013 particles. The culture supernatant is collected, and phage precipitated by the addition of 1/10 volume 20% (w/v) polyethylene glycol (PEG) 8000/ 2.5 M NaC1 (60 min, 4°). The phage are collected by centrifugation (10,000 rpm, 10 min, 4°) resuspended in 1 ml PBS containing 1% (w/v) BSA (the BSA serves to help block nonspecific binding), and filtered (0.45/xm) to remove any residual bacteria and aggregates of phage. Disk membranes containing 200/xg of phosphorylated rhodopsin 19 are added to the phage, exposed to white light (2 min) to photoactivate the RP, and allowed to incubate with gentle mixing (60 min, 4°). The membrane/phage mixture is centrifuged (16,000g, 10 min, 4°), and the pellet resuspended in 1 ml PBS containing 0.1% (v/v) Tween 20. This washing with detergent is repeated once more, and then twice more with PBS without detergent. Following the final wash, the phage are acid eluted with 400 tzl PBS, pH 4.0 containing 0.5 M NaCI (15 min at room temperature), then neutralized with 400/zl PBS, pH 11.0. The eluted phage are mixed with 800/xl 2× YT with 2% BSA, to which 200/xg RP is added and the panning process repeated. Following each panning cycle, an aliquot of the phage is used to infect TG1 E. coli cells to monitor the number of phage eluted with each cycle. For titration, TG1 cells are grown to OD600nm of 0.3, a serial titration of phage added (usually 10 1-10-5), and the infected cells plated on 2× YT agar plates with 2% glucose and 50/xg/ml carbenicillin. Each panning cycle should give approximately 102- to 103-fold enrichment of phage that bind to the substrate. If the number of infectious phage particles drops below 104, the phage should be reamplified as in step 1 before proceeding to the next cycle of panning. After completing four panning cycles, isolated colonies from the plates are cultured and phagemid DNA prepared following standard minipreparation protocols for the isolation of plasmid DNA (e.g., Wizard Miniprep kits, Promega, Madison, WI). Because the TGI strain of E. coli is not an endonuclease-deficient strain ( E n d A ) ,
~9j. Puig, A. Arendt, F. L. Tomson, G. Abdulaeva, R. Miller, P. A. Hargrave, and J. H. McDowell, FEBS Lett. 362, 185 (1995).
448
PROTEINS THAT INTERACT WITH RHODOPSIN
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proteases should be included in the sample preparation buffers in order to improve the quality of D N A for sequencing. We sequenced the phagemid D N A manually using the dideoxy termination method 2° in the presence of [35S]dATP,although automatic sequencing works as well. It is sufficient to sequence across the insertion site from both directions to determine which portion of the arrestin cDNA was cloned into the genelII D N A and to ensure that proper frame orientation is maintained. Any inserts that are not in frame with the genelII open reading frame should be discarded from the data. If only a few colonies are obtained after panning, it is an easy matter to sequence all clones. However, if several thousand colonies are obtained, then only a sample of the colonies can be reasonably sequenced. We obtained 2.4 × 103 colonies from phage panned against R*P in disk membranes and 300 colonies from phage panned against red blood cell membranes. We sequence 200 colonies from the phage panned against R*P and 50 colonies from phage panned against our control membranes. Control Considerations
In our experiment rhodopsin was provided in disk membranes prepared from rod outer segments. This provided the added component of the lipid bilayer in which the rhodopsin was embedded. The best control would have been the same lipid membrane with rhodopsin removed. However, this material is quite difficult to prepare in the quantities required for the panning experiments. Instead, we chose an alternative approach in which membranes were collected from lysed red blood cells. 21 These membranes were used in a parallel panning experiment. From the small number of colonies obtained in this control experiment we were able to conclude that the contribution due to nonspecific binding to membranes was likely to be insignificant. Data Analysis General Considerations
Data analysis is relatively straightforward since it involves simply a compilation and alignment of the sequences obtained that bound to the substrate. For random peptide sequences, the goal is to identify one or a few residues that emerge as a consensus residue in the same location of 20 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 21 D. Hanahan and J. Ekholm, Methods Enzymol. 3L 168 (1974).
[30]
MAPPING INTERACTION
SITES
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the peptide (usually present in 50-70% of the isolated peptides). For screens of a specific protein, the goal is to isolate the minimal epitope from the overlapping peptides.
Specific Application Figure 3 shows a compilation of the arrestin sequences that bound to R*P in disk m e m b r a n e s (Fig. 3A) and those that b o u n d to red blood cell m e m b r a n e s (Fig. 3B). U n d e r ideal circumstances, a minimal epitope will be revealed that can be used for further studies. In our experiment, the entire molecule of the protein was represented. Perhaps this reflects the fact that multiple portions of arrestin contribute to the binding to rhodopsin.
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However, if these data are converted to a frequency histogram (Fig. 4), simply calculating the number of times any particular amino acid of arrestin was present in a phage that bound to R'P, it is clear the most prevalent epitope was 90-140, with additional peaks at 160-210 and 240-270. Note that for the primary peak of binding (90-140) there was virtually no binding to red blood cell membranes, indicating that it is unlikely that the phage
[30]
MAPPING INTERACTION SITES
451
displaying this region of arrestin were binding nonspecifically to the lipid membrane. Data Confirmation General Considerations One critical feature of phage display is that like other library screening techniques, such as yeast two-hybrid or cDNA expression library screening, all potential positives must be tested to eliminate false-positives and establish biological relevance. This can be approached using any number of methods that are appropriate for the particular system, but usually include heterologous expression to produce protein for measuring binding parameters, receptor activation/inactivation assays, or binding competition assays. Specific Application For our arrestin phage display, we chose to express a portion of the principal binding region as a fusion with glutathione S-transferase and demonstrate that this fusion could function as a competitor with arrestin for binding to R*P. We first expressed the region of arrestin spanning amino acids 95-140 as a carboxyl-terminal fusion protein with glutathione S-transferase. The arrestin cDNA corresponding to this region was amplified by PCR, adding 5' EcoRI and 3' Sall restriction sites with synthetic oligonucleotide primers, and cloned into the pGEX-4T-1 vector (Pharmacia) at the EcoRI and SalI sites. This recombinant plasmid was then introduced into BL21 E. coli cells and used to express soluble protein, inducing with 1 mM IPTG (4 hr, 37°). The expressed protein was purified over glutathione-agarose (Pharmacia) following the manufacturer's recommended procedures. We were able to obtain approximately 2 rag/liter of pure protein. We then assayed the ability of this protein to compete for the binding of arrestin to R*P. Arrestin (10/~g) was mixed with R*P (40 ~g) contained in disk membranes in PBS in the presence of increasing amounts (0-1 raM) of 95-140/GST fusion protein, and 1/.~M glyceraldehyde 3-phosphate dehydrogenase in a 150-/~1 reaction volume. (The dehydrogenase serves as an internal control for quantitation.) Following incubation (2 rain, 4°), the reaction mixture was centrifuged (24,000g, 30 min, 4°) to sediment the disk membranes along with any bound arrestin. The amount of arrestin and R*P used was empirically determined to give complete binding of the arrestin in the absence of fusion protein. The reaction supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel
452
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Peptide Concentration ( tM) FI~. 5. Binding inhibition of arrestin to R*P using 95-140/GST fusion protein. Incubations containing 1.4 ~M arrestin, 8/~M R*P in disk membranes, and the indicated amount of fusion protein (open squares) or glutathione S-transferase alone (closed circles) in the presence of 1 t~M glyceraldehyde 3-phosphate dehydrogenase were prepared as described. The amount of arrestin competitively released into the supernatant was measured by SDS-PAGE, stained with Coomassie blue, and quantified by scanning densitometry. The curves represent the means (+ S.D.) from three experiments.
(SDS-PAGE) electrophoresis to determine if any arrestin was displaced by competition into this fraction. The amount of arrestin present in the reaction supernatant was quantified by scanning densitometry (versions of NIH Image are available for free downloading from NIH's website, http://rsb.info.nih.gov/nih-image/), normalizing the samples to the internal glyceraldehyde 3-phosphate control. Figure 5 shows that as the concentration of 95-140/GST is increased, there is an increasing amount of unbound arrestin. Note that GST alone has no influence on the binding of arrestin to R*P. If this competition is projected, the IC50 for the inhibition is projected to be in the low millimolar range. This experiment provides corroboration of the data obtained using phage display.
[30]
MAPPING INTERACTION SITES
453
Use of Synthetic Peptides Protein-protein interactions form the basis of many key events in biological processes. It is often possible to test synthetic peptides from the sequence of one of the interacting proteins to determine whether they compete in a protein-protein binding reaction, and thus determine the sites of interaction between the proteins. This technique has been successfully applied in several studies of protein interaction of interest in the study of the biochemistry of vision. Some peptides representing sequences displayed on the cytoplasmic surface of rhodopsin compete for the binding of transducin to R*, thus helping delineate binding sites on rhodopsin for transducin. 22 Synthetic peptides corresponding to two regions near the carboxyl terminus of the transducin a subunit compete with transducin for the binding to R*, thus suggesting part of the regions of transduein that interact with R*. 23 The third cytoplasmic loop peptide of rhodopsin competes for the binding of arrestin to R'P, suggesting this loop as a major site of interaction between the two proteins, a4 And the interaction site by which guanylate cyclase becomes activated by its activating protein has been shown to be within the catalytic domain of guanylate cyclase, by peptide competition. 25 Synthetic peptides have also been used to map epitope specificities of libraries of anti-rhodopsin monoclonal antibodies. 26'2v
Specific Application For this research overlapping peptides 20 amino acids in length were synthesized using RAMPS Multiple Peptide Synthesis System (DuPont, Wilmington, DE). Most peptides were synthesized using "Wang" Carboxylate Resin cartridges with 0.1 mmol of first 9-fluoroenylmethoxycarbonyl amino acids on p-alkoxybenzyl alcohol resin (DuPont). Sequential amino acids were coupled according to the RAMPS protocol using HOBt esters prepared in situ from Fmoc amino acids. Cleavage and deprotection from resin was carried out by incubating the resin for 3 hr at room temperature 22 B. K6nig, A. Arendt, J. H. McDowell, M. Kahlert, P. A. Hargrave, and K. P. Hofmann, Proc. Natl. Acad. Sci. U.S.A. 86, 6878 (1989). 23 H. E. Hamm, D. Deretic, A. Arendt, P. A. Hargrave, B. Koenig, and K. P. Hofmann, Science 241, 832 (1988). 24 j. G. Krupnick, V. V. Gurevich, T. Schepers, H. E. Hamm, and J. L. Benovic, J. Biol. Chem. 269, 3226 (1994). 25 I. Sokal, F. Haeseleer, A. Arendt, E. T. Adman, P. A. Hargrave, and K. Palczewski, Biochemistry 38, 1387 (1999). 26 R. S. Hodges, R. J. Heaton, J. M. R. Parker, L. Molday, and R. S. Molday, J. Biol. Chem. 263, 11768 (1988). 27 G. Adamus, Z. S. Zam, A. Arendt, K. Palczewski, J. H. McDowell, and P. A. Hargrave, Vis. Res. 31, 17 (1991).
454
PROTEINS THAT INTERACT WITH RHODOPSIN
[30]
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F~o. 6. Binding competition of arrestin to phosphorylated photoactivated rhodopsin using synthetic arrestin peptides. Photoactivated disk membranes containing 8/xM R*P were incubated with 1.4/zM arrestin in the presence of 700/~M syntheticpeptides. Followingcentrifugation, inhibition of arrestin bindingwas measured by analyzingthe amount of arrestin remaining in the supernatant using SDS-PAGE. Arrestin was quantified by scanning densitometry, normalizing arrestin to an internal standard (glyceraldehyde 3-phosphate dehydrogenase). The means (-+ S.D.) from three experiments are shown. in 90: 5 : 5 (v/v) trifluoroacetic acid (TFA) : H 2 0 : ethanedithiol. For purification, peptides were precipitated from solution using cold diethyl ether, lyophilized, and further purified over Bio-Gel P-2 (Bio-Rad, Hercules, CA) in 5% (v/v) acetic acid. Quality was assessed by amino acid analyses and analytical HPLC. The peptides synthesized corresponded to arrestin sequence 81-100, 91-110, 101-120, 111-130, 120-140, 131-150, 141-160, and 151-170. The peptides were tested in a binding competition assay, using the same methodology as delineated earlier for testing the effect of 95-140/GST fusion on arrestin binding to R*P except that increasing concentrations of peptides (0-700 /zM) were used instead of the fusion protein. Figure 6 shows the amount of arrestin binding that was inhibited by each peptide. These results show some effect on binding by peptides 101-120, 111-130,
[311
STRUCTURE-FUNCTION
ANALYSISOF RanBP2
455
and 121-140, with the largest effect by 111-130. These data clearly implicate the region 101-130 as containing a binding domain, which corresponds nicely with the phage display data. Because 111-130 has limited solubility in aqueous buffers, this peptide was resynthesized to include Lys-109 and Lys-ll0, as well as to substitute Cys-128 with serine (i.e., 109-130/Cys128 Ser). Using this peptide, we were able to completely block the binding of arrestin to R*P with an ICs0 of 1.1 mM. The relatively high IC50 likely indicates that arrestin has multiple points of contact with R ' P , which is consistent with the observations from other laboratories. 28-3° Note that a peptide containing the same amino acid composition, but ordered randomly (i.e., KDYLVPLSSNAPLYLGTFPFTK) was not an effective competitor. In conclusion, the use of synthetic peptides identifies a binding region in arrestin for R*P that is composed of, at least in part, amino acids 111-130. These data are consistent with those obtained using phage display. Consequently, the synthetic peptides are useful as additional corroboration of the phage display study, but also provide a more narrow definition of the binding region. Acknowledgment This research was supported by a Career D e v e l o p m e n t Award from the Research to Prevent Blindness (RPB) Foundation to W.C.S., a Senior Scientific Investigator award from R P B to P A H , grants from the National Eye Institute (EY06225, EY06226, EY0857l). and an unrestricted grant to the D e p a r t m e n t of O p h t h a l m o l o g y from RPB. _,s V. V. Gurevich and J. L. Benovic, J. BioL Chem. 268, 11628 (1993). 29 V. V. Gurevich and J. L. Benovic, J. Biol. Chem., 270, 6010 (1995). 3~ K. Palczewski, J. Buczylko, N. R. Imami, J. H. McDowell, and P. A. Hargrave, J. Biol. Chem. 266, 15334 (1991).
[31] C h a r a c t e r i z a t i o n o f R a n B P 2 - A s s o c i a t e d Components in Neuroretina
Molecular
By PAULO A. FERREIRA Introduction In search of proteins that mediate the biogenesis of vertebrate Gprotein-coupled light receptors, opsins, we have cloned several products I I p. Ferreira, J. H o m , and W. Pak, J. BioL Chem. 270, 23179 (1995).
METHODS IN ENZYMOLOGY,VOL. 315
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