[53]
BIOTINYLATED
CALMODULIN
DERIVATIVES
451
photoreactive biotin labeling of DNA probes and observed signal enhancement by iodopheno124 suggest that the above objective will be reached in the near future. 24 M. M. L. Leong and G. R. Fox, Anal. Biochem. 172, 145 (1988).
[53] Identification of C a l m o d u l i n - B i n d i n g P r o t e i n s B y MELVIN L. BILLINGSLEY, JOSEPH W. POLLI, KEITH R. PENNYPACKER, and RANDALL L. KINEAID
Introduction
The use of chemically modified proteins has proved indispensible for the elucidation of structural and functional characteristics of a wide range of proteins. Chemical perturbations of primary amino acid structure have been used (1) to investigate functional changes brought about by the perturbation and (2) to " m a p " the structural location of functional contacts between two interacting proteins? Experimentally, there are three possible consequences of such a modification. First, the functional interaction can be totally destroyed by the chemical modification. Second, the functional interaction can be unperturbed by the chemical modification. Third, the functional interaction can be partially affected by the structural modification. When designing probes for detection of protein-protein interactions, the goal is to use a chemical modification that does not affect the functional interaction; for site-specific chemical modifications for determining functional contact points, the goal is to produce a range of perturbations that block, partially block, or leave unimpaired the ability of the modified protein to interact with its putative target. Calmodulin is the major intracellular receptor for calcium; the primary and tertiary structures of the molecule have been well described. 2,3 On binding up to 4 mol of calcium/mol protein, the molecule undergoes a marked conformational change, exposing an hydrophobic a-helical region of the protein. The specificity of the Ca2+-regulated event is determined by the Ca2+-dependent interaction of this domain on calmodulin with a target binding protein. Thus, many of the varied Ca2÷-regulated cellular G. K. Ackers and F. R. Smith, Annu. Rev. Biochem. 54, 597 (1985) 2 j. C. Stoclet, D. Gerard, M.-C. Kilhoffer, C. Lugnier, R. Miller, and P. Schaffer, Prog. Neurobiol. 29, 321 (1987). 3 y. S. Babu, C. E. Bugg, and W. J. Cook, this series, Vol. 139, p. 632.
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
452
APPLICATIONS
[53]
events are ultimately mediated through the actions of calmodulin-binding proteins. 4 One approach to characterize cellular calmodulin-binding proteins is to chemically modify calmodulin with fluorescent, radioactive, or other chemically reactive congeners) ,6 Such modified calmodulins have been exploited to study conformational changes on Ca2+-dependent binding, to monitor the distribution of calmodulin in the cell, and to identify calmodulin-binding proteins following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 7-9 The use of an iodinated calmodulin gel overlay for the purpose of detecting calmodulin-binding proteins was first described by Carlin et al. lo and Glenny et al. l l In this procedure, putative calmodulin-binding proteins were subjected to SDS-PAGE and renatured in situ. The gel was incubated with trace amounts of ~25I-labeled calmodulin, washed extensively in Ca2+-containing buffers (2-3 days), and dried and subjected to autoradiography. This procedure demonstrated that calmodulin-binding proteins could be resolved on SDS-PAGE, renatured, and that the 125I-labeled calmodulin could interact with specific calmodulin-binding domains in a Ca2+-dependent manner. The gel-overlay technique has been refined to permit detection and quantification of microgram quantities of calmodulin-binding proteins. ~2 There are several drawbacks to the gel-overlay technique, particularly regarding the overall time of the procedure (7-14 days of sample preparation, washing, and autoradiography) and the amount (liters) of low-level radioactive waste generated during washing. Flanagan and Yost described a modified procedure for detection of calmodulin-binding proteins using nitrocellulose blots (Western blots) of SDS-PAGE-resolved calmodulin-binding proteins.~3 By using Tween 20 to minimize the nonspecific binding on the blot, they were able to identify calmodulin-binding proteins from both one- and two-dimensional SDS-PAGE. Furthermore, they reduced the lengthy washing times needed to reduce background in the gel-overlay protocol. This method still required the use of ~25I-labeled 4 C. B. Klee and T. C. Vanaman, Adv. Protein Chem. 35, 213 (1982). R. L. Kincaid, M. L. Billingsley, and M. Vaughan, this series, Vol. 159, p. 605. 6 T. J. Andreason, C. H. Keller, D. C. LaPorte, A. M. Edelmon, and D. R. Storm, Proc. Natl. Acad. Sci. U.S.A. 78, 2782 (1981). 7 R. L. Kincaid, M. C. Vaughan, J. C. Osborne, Jr., and V. A. Tkachuk, J. Biol. Chem. 257, 10638 (1982). 8 H. W. Jarrett, J. Biol. Chem. 261, 4967 (1986). 9 K. Luby-Phelps, F. Lanni, and D. L. Taylor, J. Cell Biol. 101, 1245 (1985). 10 R. K. Carlin, D. J. Corab, and P. Siekevitz, J. Cell Biol. 89, 449 (1981). II j. R. Glenny and K. Weber, J. Biol. Chem. 255~ 10551 (1980). 12 G. R. Slaughter and A. R. Means, this series, Vol. 139, p. 433. t3 S. D. Flanagan and B. Yost, Anal. Biochem. 140, 510 (1984).
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
453
calmodulin and subsequent autoradiography for detection of the calmodulin-binding proteins. In order to facilitate the rapid, nonradioactive detection of immobilized calmodulin-binding proteins, we devised a method for incorporating biotin into lysine residues of calmodulin.~4 By using biotin as a reporter ligand, avidin-linked enzyme systems could be used to detect biotinylated calmodulin (Bio-CaM) bound to renatured calmodulin-binding proteins. Of note is the caveat that not all proteins may "renature" following SDSPAGE and blotting; hence, care should be exercised when interpreting negative results. In addition, biotinylation can be used to explore what portion(s) of the calmodulin molecule is important for interactions with target proteins. By observing how the modification of a specific residue affects subsequent calmodulin-enzyme interactions, one can determine whether biotinylation perturbs focal points of protein-protein interaction. If biotinylation of a specific residue does not affect calmodulin interaction directly, one can preincubate biotinylated calmodulin derivatives with avidin, and determine whether complexing biotinylated calmodulin with avidin can block subsequent binding or enzymatic activation. 15However, the avidin-biotinylated calmodulin complexes may sterically or allosterically interfere with the calmodulin-binding protein interaction and, thus, cannot be used to directly prove that a particular site is necessary for interaction. Biotinylated calmodulin can be used to study differential expression of calmodulin-binding proteins in tissues or cell populations. We have exploited this technique to elucidate the major calmodulin-binding proteins of lymphocytes. In addition, biotinylated calmodulin can also be used to identify specific calmodulin-binding peptides following proteolytic degradation of the intact protein; comparison of such "fingerprints" may indicate whether they are structural features common to a series of calmodulin-binding proteins. Finally, Bio-CaM may prove useful for microinjection studies, since the biotin reporter group can be easily localized in cells using avidin-based histochemistry. However, careful titration of injected Bio-CaM would be needed in order to avoid detection of unbound Bio-CaM. One key point is that in order to detect the interaction between calmodulin and specific calmodulin-binding proteins, one must generate a chemically modified probe that is functionally competent. Owing to the high methionine content (8 residues) of calmodulin, oxidative approaches to 14 M. L. Billingsley, K. R. Pennypacker, C. G. Hoover, D. J. Brigati, and R. L. Kincaid, Proc. Natl. Acad. Sci. U.S.A. 82, 7585 (1985). 15 D. Mann and T. C. Vanaman, this series, Vol. 139, p. 417.
454
APPLICATIONS
[53]
modification often alter biological activity. 14,15 In addition, there are no cysteine residues, further narrowing the choice of sites for modification. Thus, numerous studies have focused on amine-directed agents for modification. However, as several studies have indicated, the 7 lysyl residues in calmodulin differ with respect to their relative reactivities; depending on the target protein, several of the lysines may be critical for calmodulintarget interactions.16-19 Since many lysines may be modified during reaction, such "derivatives" are not homogeneous populations; thus, caution must be used when interpreting the results obtained with modified molecules. One empirical approach is to produce a series of biotinylated calmodulins modified at lysyl residues (using N-hydroxysuccinimide esters), at tyrosyl or histidyl residues (using p-diazobenzoylbiocytin), at acidic amino acid residues (using biotin hydrazide and carbodiimide coupling), or at nondirected, accessible nucleophilic sites (using "photobiotin" acetate). Modification of acidic amino acids in calmodulin with biotin hydrazide has proved to be of limited utility in our hands and is not described in detail. Preparation of Biotinylated Calmodulin Derivatives We have described the preparation and uses of calmodulin modified using biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester in several prior publications, and readers are referred to Refs. 5, 14, and 20 for specific modification. In this chapter, we focus on the reagents needed for producing other forms of biotinylated calmodulin. All modifications are carried out in the presence of 1 mM CaCI2 in order to ensure modification of a homogeneous population of fully liganded proteins, i.e., to avoid partially or nonliganded forms.
Reagents Purified bovine brain or testis calmodulin, 2 mg/ml, in coupling buffer [0.1 M sodium phosphate buffer (pH 7.4) or 0.1 M HEPES (pH 7.0)] 21 16 D. P. Giedroc, S. K. Sinha, K. Brew, and D. Puett, J. Biol. Chem. 260, 13406 (1985). IT D. P. Giedroc, D. Puett, S. K. Sinha, and K. Brew, Arch. Biochem. Biophys. 252, 136 (1987). is F. M. Faust, M. Slisz, and H. W. Jarret, J. Biol. Chem. 262, 1938 (1987). 19 M. A. Winkler, V. A. Fried, D. L. Merat, and W. Y. Cheung, J. Biol. Chem. 262, 15466 (1987). 2o M. L. Billingsley, K. R. Pennypacker, C. G. Hoover, and R. L. Kincaid, BioTechniques 5, 22 (1987). 21 R. L. Kincaid, this series, Vol. 139, 3 (1987).
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
455
0.1 M CaCl2 0.1 M EGTA 1.0 M NaCl NHS-LC-biotin (Pierce Chemical Co., Rockford, IL), 26 mg/ml in dry N,N-dimethylformamide (10:1 molar ratio of derivative to calmodulin) Photobiotin acetate (Research Organics, Cleveland, OH), 2 mg/ml in coupling buffer (10:1 molar ratio of derivative to calmodulin) Sulfo-NHS-biotin (Pierce), 21 mg/ml in dry N,N-dimethylformamide (10 : 1 molar ratio of derivative to calmodulin) Activated diazobenzoylbiocytin (Calbiochem, La Jolla, CA), see below for method of activation Nitrocellulose membrane (BA-85, Schleicher & Schuell, Keene, NH) Blocking solution [50 mM Tris-HCl (pH 7.4) containing 150 mM NaC1, 1 mM CaCI2, 0.1% antifoam A (Sigma Chemical Co., St. Louis, MO), and 5% nonfat dry milk] Wash buffer [50 mM Tris-HC1 (pH 7.4) containing 1 mM CaC12 and 150 mM NaCI] Avidin-alkaline phosphatase or avidin-horseradish peroxidase detection systems (Vector Laboratories, Burlingame, CA; BioMeda, Inc.) Hydrogen peroxide, 30% stock solution (v/v) p-Chloronaphthol (Sigma), 1 mg/ml, in wash buffer containing 20% methanol (v/v) 5-Bromo-4-chloro-3-indoyl phosphate p-toluidine (BCIP; Amresco), 50 mg/ml, in N,N-dimethylformamide Nitroblue tetrazolium chloride (NBT; Amresco), 50 mg/ml, in 50% N,N-dimethylformamide Phosphatase buffer [0.1 M Tris-HCl (pH 9.5) containing 100 mM NaC1 and 50 mM MgCI2] Avidin (Sigma or other source) d-Biotin (Sigma or Pierce) Dialysis membrane (Spectrapor; 6,000 MW cutoff)
Preparation of Sulfo-NHS-Biotin- and NHS-LC-Biotin-Derivatized Calmodulin Purified calmodulin, prepared using melittin-Sepharose, 2~ is dialyzed against coupling buffer, and the concentration is adjusted to 1.0 mg/ml (~.1% ~,280 = 1.8). A 2-rag aliquot of calmodulin solution is adjusted to 1 mM CaCI2 and 0.15 M NaCI. An aliquot (25/.d) of either NHS-LC-biotin (0.66 rag/25/A) or sulfo-NHS- biotin (0.53 mg/25/~1) is added at a 10-fold molar
456
APPLICATIONS
[53]
excess with respect to calmodulin. Previous studies have indicated that the 10:1 molar excess of NHS derivative gives optimal labeling with minimal loss of biological function. 2° The derivatization is carried out for 2 hr at 25 °, and the mixture is then either desalted on a precalibrated Sephadex G-25 column (PD-10, Pharmacia) or dialyzed exhaustively against coupling buffer to remove unreacted reagent. The calmodulin derivatives are stored in coupling buffer with 10% glycerol at - 2 0 °.
Preparation of Photobiotin Acetate-Derivatized Calmodulin Purified calmodulin (in 1 mM CaCI2 and 150 mM NaCI) is dialyzed as described above. In subdued light, photobiotin acetate (0.71 mg/335 ~1) is dissolved in water and added to the calmodulin solution (2 mg total) in a fiat-bottomed tube kept at 4 ° using the precautions and procedures previously outlined. 22 The reaction mixture is then placed 10 cm from a broadspectrum GE sunlamp and irradiated for 10 min at 4°. The reaction mixture is either dialyzed against coupling buffer or chromatographed over a PD-10 Sephadex column to remove unreacted photobiotin. The derivatized calmodulin is stored in coupling buffer containing 10% glycerol at - 2 0 °"
Preparation of p-Diazobenzoylbiocytin-Derivatized Calmodulin p-Diazobenzoylbiocytin (DBB) reagent is activated as previously described23:2 mg of DBB is dissolved in 15/zl of dimethyl sulfoxide. Eightyfive microliters of 1 M HC1 is added, and the mixture is chilled on ice. To this mixture, 40/zl of0.112 M sodium nitrate is added and allowed to react for 5 min. This activation is quenched by adding 100/xl of 1 M NaOH, and the reaction is diluted with 1.75 ml of coupling buffer. In order to start the modification, 1 ml of purified, dialyzed calmodulin (2 mg/ml coupling buffer, brought to 1 m M CaC12and 150 mM NaC1) is reacted with 500/zl of the activated DBB mixture for 1.5 hr at 25°. Following incubation, the reactants are removed by chromatography over PD-10 Sephadex G-25 columns. The derivative is stored in coupling buffer containing 10% glycerol at - 2 0 ° . Characterization of Biotinylated Calmodulin Derivatives A useful method for documenting the production of the various biotinylated species is to determine the ultraviolet spectrum of the calmo22 E. Lacey and W. N. Grant, Anal. Biochem. 163, 151 (1987). 23 M. Wilchek, H. Ben-Hur, and E. A. Bayer, Biochem. Biophys. Res. Commun. 138, 872 (1986).
[53]
457
BIOTINYLATED CALMODULIN DERIVATIVES
dulin before and after derivatization. As can be seen in Fig. 1A-C, all of the modified derivatives result in spectral shifts relative to native calmodulin. Sulfo-NHS-biotin and NHS-LC-biotin derivatives (data not shown) give nearly identical spectra (Fig. 1A), with absorbance maxima shifted from 277 nm (native calmodulin) to 266 nm (NHS-modified calmodulin). Thus, there is no apparent difference in the extent or amount of labeling of calmodulin produced by the more water-soluble sulfo-NHS-biotin reagent or by the more hydrophobic NHS-LC-biotin derivative. Photobiotin (Fig. 1B) and DBB (Fig. 1C) produce a different type of spectral shift, with DBB-modified biotin exhibiting considerable absorbance between 320 and 260 nm. Thus, different chemistries of derivatization result in characteristic spectral patterns of the modified calmodulins. A second method for demonstrating biotinylation is direct visualization of biotinylated calmodulins following transfer to nitrocellulose. Although calmodulin has been reported to bind poorly to nitrocellulose, 24we have found that calmodulin easily and rapidly transfers from 20% polyacrylamide gels to nitrocellulose, provided that the blot is transferred for 1.0
A
U.I 0
Z
.,,,,,.SULFO-BNHS
n." 0
co
NATIVE CaM
0
I
220
I
244
l
I
I
268
I
292
I
~
316
J
340
nm FIG. 1. UV absorbance spectra of biotin-labeled calmodulin preparations. (A) Native calmodulin and NHS-LC-biotin-derivatized calmodulin; (B) photobiotin-modified calmodulin; and (C) DBB-modified calmodulin. All solutions were approximately 2 mg/ml calmodulin in coupling buffer (pH 7.0) and were scanned using a Beckman DU-7 scanning spectrophotometer.
1.0
B
14.1 (..) Z
PHOTOBIOTIN
_~
0
NATIVE CaM
,
'
0 220
244
268
292
316
~40
nm 1.0
C
LU Z
rn nO
NATIV E ~ CaM
0
"'"J
I
220
244
I
"~
L
i
268
I
292 nm
FIG. 1. (continued)
I
,
316
i
340
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
459
only 20 min at 200 mA; normal blotting times of 2-4 hr result in calmodulin passing through the nitrocellulose and into the transfer buffer. In order to further stabilize adsorption of calmodulin on nitrocellulose blots, the freshly transferred blot is immediately fixed in a solution of 10% acetic acid-25% (v/v) 2-propanol for 30 min prior to subsequent processing. Although others have shown that calmodulin will bind to nylon-based membranes, 24 we have found that most nylon membranes result in an unacceptable level of color background when incubated with avidin-based chromogenic enzymes. By reducing the time and current of cell transfer and by fixing the nitrocellulose prior to incubation, satisfactory binding and detection of biotinylated calmodulin on nitrocellulose can be achieved. NHS-LC-Biotinylated calmodulin can be easily detected following transfer to nitrocellulose. For most modifications as little as 20 mg biotinylated calmodulin can be detected. Following dialysis, either 10 or 20 /zg of native or biotinylated calmodulin is subjected to SDS-PAGE (20% gel), transferred, and fixed as described above, and nonspecific binding sites are blocked using blocking buffer for 30 min. Following 3 washes for 15 min each, the blot is incubated with avidin-peroxidase (1 /zg/ml in wash buffer) for 30 min, washed 3 additional times in wash buffer, and reacted with substrate solution, consisting of 0.03% H202 and pchloronaphthol (1 mg/ml) in wash buffer containing 20% methanol. Biotinylated calmodulin is easily visualized following this procedure, while native calmodulin does not generate a signal. Similar results can be obtained with the other calmodulin derivatives. On occasion, particularly if excess molar ratios of derivatization agent are used, several calmodulin peptides of altered electrophoretic mobility can also be visualized on blots; however, these preparations are not effective as probes for blot overlays. 2° The more slowly moving peptides most likely represent calmodulins containing multiple biotinylated amino acid residues and, possibly, having impaired Ca z+ binding, which also decreases electrophoretic mobility. In order to determine whether the biotinylated calmodulin derivatives can recognize calmodulin-binding proteins immobilized on nitrocellulose (Fig. 2), fractions of rat cerebral cortex synaptosomes (lane 1; P2 fraction), cerebral cortical cytoplasm (lane 2; $2 fraction), and partially purified rat cytosolic calmodulin-binding proteins (primarily calcineurin at 60K and caldesmon at 150K and 105K) were subjected to SDS-PAGE and electroblotted. Identical blots are incubated with the biotinylated calmodulin derivatives as follows. Following a 30-min incubation in blocking buffer, blots are washed 3 times for 10 min in wash buffer and 24 C. R. Egly and D. Daviaud, Electrophoresis 6, 325 (1985).
460
APPLICATIONS BNHS
Mr(XJ0 -3) 1
2
PHOTOBIOTIN 3
1
2
3
[53]
DBB 1
2
SULFO-NHS 3
1
2
3
CONTROL 1
2
3
FIG. 2. Relative reactivityof several biotinylatedcalmodulinderivatives. Calmodulin was modifiedwith NHS-LC-biotin(BNHS), sulfo-NHS-biotin,photobiotin, or DBB and incubatedwithblots containingcerebral cortex membranes(lane 1,100v.g),cerebralcortical cytosol (lane 2, 100/~g), or partially purified calmodulin-bindingproteins (lane 3, 10/xg). Followingincubation with biotinylatedcalmodulins,the blots were incubatedwith avidinalkaline phosphatase and developedwith appropriate chromogens.An identicalblot (Control) was reacted with avidin-alkaline phosphatase alone, revealing endogenous biotincontaining peptides having molecularweights of 75,000 and 100,000.
incubated with calmodulin biotinylated using NHS-LC-biotin (BNHS), photobiotin, DBB, or sulfo-NHS-biotin. Each congener is incubated at a concentration of 5/xg/ml in blocking buffer for 30 min, followed by 3 10-min washes in wash buffer. Avidin-alkaline phosphatase complexes (1 /zg/ml wash buffer) are then incubated with the blot for 20 min, followed by 3 additional 10-min washes. A control consists of blotted samples incubated with avidin-alkaline phosphatase alone. The avidin detection system step should not be carried out in the usual blocking buffer because of occasional contamination of the nonfat dry milk with biotin. Each blot is incubated with BCIP-NBT (100 ~1 BCIP stock, 200 t~l NBT stock in 30 ml of phosphatase buffer) until colored bands appear. The color development is carried out under subdued light and can proceed for as long as 12 hr without excessive background staining. As shown in Fig. 2, all biotinylated calmodulin derivatives bind to a number of immobilized calmodulin-binding proteins from rat brain. Some differences in binding were noted, however. NHS-LC-biotin (BNHS) and sulfo-NHS-biotin derivatives of calmodulin recognize several brain
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
461
calmodulin-binding proteins, with prominent staining of peptides at Mr 52,000, 60,000-62,000, 75,000, 85,000, and 105,000-240,000; this pattern is reminiscent of Bio-CaM used in previous studies.14 Control incubations have demonstrated that the peptides at Mr 75,000 and 100,000 bind avidin-alkaline phosphatase alone, suggesting that these peptides contain biotin. DBB-calmodulin gives a less intense pattern of labeling (see staining of the 52,000 peptide) and fails to recognize the 60K subunit of calcineurin (Lane 3). Thus, modification of histidyl and/or tyrosyl residues with DBB appears to alter the recognition of specific binding domains. This may suggest that a specific histidine or tyrosine residue is important for interaction of DBB-calmodulin with immobilized calcineurin. Studies are in progress to investigate this possibility. Additional methods for characterizing the calmodulin derivatives include tests of their ability to stimulate calmodulin-dependent enzymes, limited proteolysis of biotin-calmodulin, and direct sequencing of the modified molecule(s). Past studies have indicated that calmodulin modified with biotin-NHS derivatives retains the ability to stimulate calmodulin-dependent phosphatase activity ~4and calmodulin-dependent phosphodiesterase. 15 Interestingly, Mann and Vanaman observed that although BNHS-calmodulin could stimulate phosphodiesterase, preincubation with avidin inhibited calmodulin stimulation of phosphodiesterase activity. 15 We have observed that phosphodiesterase is poorly detected by BNHS-calmodulin on blot overlays, in agreement with the idea that the biotinylated lysine is obscured following calmodulin interaction with phosphodiesterase. Alternatively, the interaction of avidin may alter the calmodulin conformation such that interaction with phosphodiesterase cannot take place. Mann and Vanaman have sequenced BNHS-calmodulin and found that the biotin label is primarily localized on lysine-94. Thus, biotinylated calmodulin derivatives may prove quite useful in mapping points of interaction between calmodulin and its targets. Biotinylated calmodulin can be purified using a variety of chromatographic techniques. Hydrophobic interaction chromatography can be used to purify calmodulin; however, this method does not resolve biotinylated from native calmodulin. 25 HPLC using DEAE resins has been used to separate modified from native calmodulin. ~5We have used avidinagarose to purify biotinylated calmodulin; however, harsh elution conditions (pH 2.5; 6 M guanidine) must be used (M. Billingsley, unpublished observation). 2s R. Gopalakrishna and W. B. Anderson, Biochem. Biophys. Res. Commun. 104, 830 (1982).
462
APPLICATIONS
[53]
Uses of Biotinylated Calmodulin
Identification of Tissue-Specific Calmodulin-Binding Proteins The effect of Ca2÷-calmodulin in a given tissue is determined by the expression of particular calmodulin-binding proteins in the various cells of the tissue. We have used BNHS-calmodulin to determine the pattern of calmodulin-binding protein expression in various rat tissues. Membrane preparations, derived from heart, kidney, testis, lung, and pancreas, and equal amounts of tissue protein (100/xg/lane) were subjected to SDS-PAGE and transferred to nitrocellulose. As shown in Fig. 3, each tissue exhibits a distinct pattern of calmodulin-binding proteins. Although some calmodulin-binding proteins are common among tissues, others are specific to a given tissue. Two controls are essential in such studies. One is to determine the Ca 2+ specificity of the biotinylated calmodulin binding by including EGTA in an identical blot incubation (data not shown). Another complementary control is to incubate an identical blot with avidin-alkaline phosphatase alone in order to determine the extent of endogenous biotin-containing proteins in a tissue. It is also possible to block the reactivity of endogenous biotin-containing proteins by incubating the preblocked blot with avidin (0.1 mg/ml in wash buffer containing 1% bovine serum albumin) followed by a wash with excess biotin (1 mg/ml). We have found it easier to locate and "subtract" the biotin containing proteins from the calmodulin-binding proteins, in part because avidin "blocking" can give equivocal results (M. Billingsley and R. Kincaid, unpublished observation).
Detection of Reginal and Subcellular Differences in Calmodulin-Binding Protein Expression Biotinylated calmodulin overlays can also be used to explore the differences in calmodulin-binding proteins in subcellular fractions from brain regions. An example of such a separation is shown in Fig. 4. Rat brain is dissected into cerebral cortex, cerebellum, hippocampus, and striatum. Each tissue is homogenized and separated into synaptosomal (P2) and cytosolic ($2) fractions. Individual lanes of a 10% polyacrylamide gel are loaded with 75/zg total protein, and the resolved proteins are transferred to nitrocellulose. Following blocking as described above, the blot is reacted with NHS-LC-biotinylated calmodulin (5 ~g/ml blocking solution) and detected using avidin-alkaline phosphatase and BCIP-NBT chromagen systems. As shown in Fig. 4, there are marked regional and subcellular differences in calmodulin-binding proteins. Most notable is the relative absence
[53]
463
BIOTINYLATED CALMODULIN DERIVATIVES
PROTEIN STAIN
BIOCAM BLOT
Mr (Xl 0-3)
94--
(57--
43--
--61
I
30-FIc. 3. Tissue distribution of membrane-associated rat calmodulin-binding proteins. Homogenates of rat heart, kidney, testis, lung, and pancreas were centrifuged at 20,000 g and subjected to S D S - P A G E and blotting as described. The blot was incubated with NHS-LCbiotinylated calmodulin and detected using avidin-alkaline phosphatase systems. The peptide at 75K bound avidin-alkaline phosphatase alone. The 61K marker corresponds to the approximate migration of purified calcineurin.
of the 52K subunit of calmodulin-dependent protein kinase II in cerebellum (lanes 2, 6); this finding has been noted by several investigators. 26,27In addition, there are regional differences in calmodulin-binding peptides between 65K and 87K, with cerebellum exhibiting the greatest difference. The $2 fraction is relatively devoid of high molecular weight calmodulinbinding proteins (i.e., spectrin, caldesmon, and proteolytic fragments thereof), whereas the P2 fraction contains a 36K calmodulin-binding pro26 S. D. Flanagan, B. Yost, and G. Crawford, J. Cell Biol. 94, 743 (1982). 27 T. L. McGuinness, Y. Lai, and P. Greengard, J. Biol. Chem. 260, 1696 (1985).
464
APPLICATIONS
[53]
Sa rv1 r
1
2
3
4
5
6
7
8
(XlO-3)
94-
67-
43-
36FIG 4. Regional and subcellular distribution of calmodulin-binding proteins in rat cortex
(lanes l, 5), cerebellum (lanes 2, 6), hippocampus (lanes 3, 7), and striatum (lanes 4, 8). Regions were separated into crude synaptosomal (P2) and cytosolic ($2) fractions as described. The blot was incubated with NHS-LC-biotinylated calmodulin and detected using alkaline phosphatase and chromogen systems.
tein termed cytosynalin 28 not seen in the cytosolic fraction. Thus, the biotinylated calmodulin overlay can be used to study regional and subcellular differences in expression of target enzymes. Of note is that, following electrophoresis and blotting, these results are obtained within 3 hr of 28 K. Sobue, T. Okabe, K. Kadowaki, K. Itoh, T. Tanaka, and Y. Fujio, Proc. Natl. Acad. Sci. U.S.A. 84, 1916 (1987).
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
BioCaM
465
Mr (X 10-3) --94 --67
-- 26
-- 18
1
2
3
FIG. 5. Identification of calmodulin-binding peptides after limited proteolysis, SDSPAGE, and transfer to nitrocellulose. Extracts of calmodulin-Sepharose eluates of T lymphocytes (lane 1), splenocytes (lane 2), and thymocytes (lane 3) were incubated for 30 min at 37° with staphylococcal V8 protease, electrophoresed on a 15% polyacrylamide gel in the presence of SDS, and transferred to nitrocellulose. The blot was incubated with biotinylated calmodulin and detected using avidin-alkaline phosphatase systems. T lymphocytes and splenocytes contain a 59K isoform of calcineurin, whereas thymocytes contain a 65K form; digestions indicate nearly identical calmodulin-binding peptides (lanes 2 and 3) from both the 59K and 65K forms. (Adapted, with permission, from Ref. 29.) incubation; similar studies with iodinated calmodulin would take considerably longer.
Proteolytic Mapping of Calmodulin-Binding Peptides W e h a v e a l s o u s e d b i o t i n y l a t e d c a l m o d u l i n o v e r l a y s to o b t a i n " f i n g e r p r i n t s " o f c a l m o d u l i n - b i n d i n g p e p t i d e s f o l l o w i n g l i m i t e d p r o t e o l y t i c di-
466
APPLICATIONS
[53]
gestion. In a study on the characterization of calmodulin-binding proteins from murine T and B lymphocytes and thymocytes, we have observed that thymocytes contain a major 65,000 calmodulin-binding protein which reacts weakly with antibodies against calcineurin. 29 Since the 59K calmodulin-binding subunit of calcineurin is the major calmodulin-binding peptide in B and mature T lymphocytes, we have used limited proteolysis of both the 65,000 and 59,000 peptides to show substantial relatedness between the calmodulin-binding peptides of these two proteins (Fig. 5). Although this approach may not be applicable to all calmodulin-binding proteins, the results indicate an important potential use of the calmodulin blot-overlay technique. Summary We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulinbinding protein expression, physical points of calmodulin-target interaction, and proteolytic maping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of proteinprotein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, nearphysiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. ~5 One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetyla29 R. L. Kincaid, H. Takayama, M. L. Billingsley, and M. V. Sitkovsky, Nature (London) 330, 176 (1987).
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IMMUNOASSAYS: OVERVIEW
467
tion experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. 3° The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins. Acknowledgments This research was supported by a research grant from The InternationalLife Sciences Institute Research Foundation and by U.S. Public Health Service Grant R01-AG06337 to M.L.B. The authors thank Ms. Doris Lineweaverfor manuscriptpreparation. 30 A. S. Manalan and C. B. Klee, Biochemistry 26, 1382 (1987).
[54] A v i d i n - B i o t i n M e d i a t e d I m m u n o a s s a y s : O v e r v i e w
By
M E I R W I L C H E K a n d E D W A R D A . BAYER
Progress in the use of avidin-biotin technology in immunoassays has developed together with major advances of the immunodiagnostics field in general. Thus, whenever a particular improvement in a given step was desired, the applicability of the avidin-biotin complex was rapidly demonstrated. At the beginning, in the late 1960s and early 1970s, when the need for improved diagnostics was established and different approaches (e.g., radioimmunoassay1 and bacteriophage 2 assay systems for the quantification i R. S. Yalow and S. A. Berson, Nature (London) 184, 1648 (1959); D. S. Skelley, L. P. Brown, and P. K. Besch, Clin. Chem. 19, 146 (1973). 20. MS.kel~., Immunology 10, 81 (1966).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOTINYLATED
CALMODULIN
DERIVATIVES
451
photoreactive biotin labeling of DNA probes and observed signal enhancement by iodopheno124 suggest that the above objective will be reached in the near future. 24 M. M. L. Leong and G. R. Fox, Anal. Biochem. 172, 145 (1988).
[53] Identification of C a l m o d u l i n - B i n d i n g P r o t e i n s B y MELVIN L. BILLINGSLEY, JOSEPH W. POLLI, KEITH R. PENNYPACKER, and RANDALL L. KINEAID
Introduction
The use of chemically modified proteins has proved indispensible for the elucidation of structural and functional characteristics of a wide range of proteins. Chemical perturbations of primary amino acid structure have been used (1) to investigate functional changes brought about by the perturbation and (2) to " m a p " the structural location of functional contacts between two interacting proteins? Experimentally, there are three possible consequences of such a modification. First, the functional interaction can be totally destroyed by the chemical modification. Second, the functional interaction can be unperturbed by the chemical modification. Third, the functional interaction can be partially affected by the structural modification. When designing probes for detection of protein-protein interactions, the goal is to use a chemical modification that does not affect the functional interaction; for site-specific chemical modifications for determining functional contact points, the goal is to produce a range of perturbations that block, partially block, or leave unimpaired the ability of the modified protein to interact with its putative target. Calmodulin is the major intracellular receptor for calcium; the primary and tertiary structures of the molecule have been well described. 2,3 On binding up to 4 mol of calcium/mol protein, the molecule undergoes a marked conformational change, exposing an hydrophobic a-helical region of the protein. The specificity of the Ca2+-regulated event is determined by the Ca2+-dependent interaction of this domain on calmodulin with a target binding protein. Thus, many of the varied Ca2÷-regulated cellular G. K. Ackers and F. R. Smith, Annu. Rev. Biochem. 54, 597 (1985) 2 j. C. Stoclet, D. Gerard, M.-C. Kilhoffer, C. Lugnier, R. Miller, and P. Schaffer, Prog. Neurobiol. 29, 321 (1987). 3 y. S. Babu, C. E. Bugg, and W. J. Cook, this series, Vol. 139, p. 632.
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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events are ultimately mediated through the actions of calmodulin-binding proteins. 4 One approach to characterize cellular calmodulin-binding proteins is to chemically modify calmodulin with fluorescent, radioactive, or other chemically reactive congeners) ,6 Such modified calmodulins have been exploited to study conformational changes on Ca2+-dependent binding, to monitor the distribution of calmodulin in the cell, and to identify calmodulin-binding proteins following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 7-9 The use of an iodinated calmodulin gel overlay for the purpose of detecting calmodulin-binding proteins was first described by Carlin et al. lo and Glenny et al. l l In this procedure, putative calmodulin-binding proteins were subjected to SDS-PAGE and renatured in situ. The gel was incubated with trace amounts of ~25I-labeled calmodulin, washed extensively in Ca2+-containing buffers (2-3 days), and dried and subjected to autoradiography. This procedure demonstrated that calmodulin-binding proteins could be resolved on SDS-PAGE, renatured, and that the 125I-labeled calmodulin could interact with specific calmodulin-binding domains in a Ca2+-dependent manner. The gel-overlay technique has been refined to permit detection and quantification of microgram quantities of calmodulin-binding proteins. ~2 There are several drawbacks to the gel-overlay technique, particularly regarding the overall time of the procedure (7-14 days of sample preparation, washing, and autoradiography) and the amount (liters) of low-level radioactive waste generated during washing. Flanagan and Yost described a modified procedure for detection of calmodulin-binding proteins using nitrocellulose blots (Western blots) of SDS-PAGE-resolved calmodulin-binding proteins.~3 By using Tween 20 to minimize the nonspecific binding on the blot, they were able to identify calmodulin-binding proteins from both one- and two-dimensional SDS-PAGE. Furthermore, they reduced the lengthy washing times needed to reduce background in the gel-overlay protocol. This method still required the use of ~25I-labeled 4 C. B. Klee and T. C. Vanaman, Adv. Protein Chem. 35, 213 (1982). R. L. Kincaid, M. L. Billingsley, and M. Vaughan, this series, Vol. 159, p. 605. 6 T. J. Andreason, C. H. Keller, D. C. LaPorte, A. M. Edelmon, and D. R. Storm, Proc. Natl. Acad. Sci. U.S.A. 78, 2782 (1981). 7 R. L. Kincaid, M. C. Vaughan, J. C. Osborne, Jr., and V. A. Tkachuk, J. Biol. Chem. 257, 10638 (1982). 8 H. W. Jarrett, J. Biol. Chem. 261, 4967 (1986). 9 K. Luby-Phelps, F. Lanni, and D. L. Taylor, J. Cell Biol. 101, 1245 (1985). 10 R. K. Carlin, D. J. Corab, and P. Siekevitz, J. Cell Biol. 89, 449 (1981). II j. R. Glenny and K. Weber, J. Biol. Chem. 255~ 10551 (1980). 12 G. R. Slaughter and A. R. Means, this series, Vol. 139, p. 433. t3 S. D. Flanagan and B. Yost, Anal. Biochem. 140, 510 (1984).
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
453
calmodulin and subsequent autoradiography for detection of the calmodulin-binding proteins. In order to facilitate the rapid, nonradioactive detection of immobilized calmodulin-binding proteins, we devised a method for incorporating biotin into lysine residues of calmodulin.~4 By using biotin as a reporter ligand, avidin-linked enzyme systems could be used to detect biotinylated calmodulin (Bio-CaM) bound to renatured calmodulin-binding proteins. Of note is the caveat that not all proteins may "renature" following SDSPAGE and blotting; hence, care should be exercised when interpreting negative results. In addition, biotinylation can be used to explore what portion(s) of the calmodulin molecule is important for interactions with target proteins. By observing how the modification of a specific residue affects subsequent calmodulin-enzyme interactions, one can determine whether biotinylation perturbs focal points of protein-protein interaction. If biotinylation of a specific residue does not affect calmodulin interaction directly, one can preincubate biotinylated calmodulin derivatives with avidin, and determine whether complexing biotinylated calmodulin with avidin can block subsequent binding or enzymatic activation. 15However, the avidin-biotinylated calmodulin complexes may sterically or allosterically interfere with the calmodulin-binding protein interaction and, thus, cannot be used to directly prove that a particular site is necessary for interaction. Biotinylated calmodulin can be used to study differential expression of calmodulin-binding proteins in tissues or cell populations. We have exploited this technique to elucidate the major calmodulin-binding proteins of lymphocytes. In addition, biotinylated calmodulin can also be used to identify specific calmodulin-binding peptides following proteolytic degradation of the intact protein; comparison of such "fingerprints" may indicate whether they are structural features common to a series of calmodulin-binding proteins. Finally, Bio-CaM may prove useful for microinjection studies, since the biotin reporter group can be easily localized in cells using avidin-based histochemistry. However, careful titration of injected Bio-CaM would be needed in order to avoid detection of unbound Bio-CaM. One key point is that in order to detect the interaction between calmodulin and specific calmodulin-binding proteins, one must generate a chemically modified probe that is functionally competent. Owing to the high methionine content (8 residues) of calmodulin, oxidative approaches to 14 M. L. Billingsley, K. R. Pennypacker, C. G. Hoover, D. J. Brigati, and R. L. Kincaid, Proc. Natl. Acad. Sci. U.S.A. 82, 7585 (1985). 15 D. Mann and T. C. Vanaman, this series, Vol. 139, p. 417.
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modification often alter biological activity. 14,15 In addition, there are no cysteine residues, further narrowing the choice of sites for modification. Thus, numerous studies have focused on amine-directed agents for modification. However, as several studies have indicated, the 7 lysyl residues in calmodulin differ with respect to their relative reactivities; depending on the target protein, several of the lysines may be critical for calmodulintarget interactions.16-19 Since many lysines may be modified during reaction, such "derivatives" are not homogeneous populations; thus, caution must be used when interpreting the results obtained with modified molecules. One empirical approach is to produce a series of biotinylated calmodulins modified at lysyl residues (using N-hydroxysuccinimide esters), at tyrosyl or histidyl residues (using p-diazobenzoylbiocytin), at acidic amino acid residues (using biotin hydrazide and carbodiimide coupling), or at nondirected, accessible nucleophilic sites (using "photobiotin" acetate). Modification of acidic amino acids in calmodulin with biotin hydrazide has proved to be of limited utility in our hands and is not described in detail. Preparation of Biotinylated Calmodulin Derivatives We have described the preparation and uses of calmodulin modified using biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester in several prior publications, and readers are referred to Refs. 5, 14, and 20 for specific modification. In this chapter, we focus on the reagents needed for producing other forms of biotinylated calmodulin. All modifications are carried out in the presence of 1 mM CaCI2 in order to ensure modification of a homogeneous population of fully liganded proteins, i.e., to avoid partially or nonliganded forms.
Reagents Purified bovine brain or testis calmodulin, 2 mg/ml, in coupling buffer [0.1 M sodium phosphate buffer (pH 7.4) or 0.1 M HEPES (pH 7.0)] 21 16 D. P. Giedroc, S. K. Sinha, K. Brew, and D. Puett, J. Biol. Chem. 260, 13406 (1985). IT D. P. Giedroc, D. Puett, S. K. Sinha, and K. Brew, Arch. Biochem. Biophys. 252, 136 (1987). is F. M. Faust, M. Slisz, and H. W. Jarret, J. Biol. Chem. 262, 1938 (1987). 19 M. A. Winkler, V. A. Fried, D. L. Merat, and W. Y. Cheung, J. Biol. Chem. 262, 15466 (1987). 2o M. L. Billingsley, K. R. Pennypacker, C. G. Hoover, and R. L. Kincaid, BioTechniques 5, 22 (1987). 21 R. L. Kincaid, this series, Vol. 139, 3 (1987).
[53]
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0.1 M CaCl2 0.1 M EGTA 1.0 M NaCl NHS-LC-biotin (Pierce Chemical Co., Rockford, IL), 26 mg/ml in dry N,N-dimethylformamide (10:1 molar ratio of derivative to calmodulin) Photobiotin acetate (Research Organics, Cleveland, OH), 2 mg/ml in coupling buffer (10:1 molar ratio of derivative to calmodulin) Sulfo-NHS-biotin (Pierce), 21 mg/ml in dry N,N-dimethylformamide (10 : 1 molar ratio of derivative to calmodulin) Activated diazobenzoylbiocytin (Calbiochem, La Jolla, CA), see below for method of activation Nitrocellulose membrane (BA-85, Schleicher & Schuell, Keene, NH) Blocking solution [50 mM Tris-HCl (pH 7.4) containing 150 mM NaC1, 1 mM CaCI2, 0.1% antifoam A (Sigma Chemical Co., St. Louis, MO), and 5% nonfat dry milk] Wash buffer [50 mM Tris-HC1 (pH 7.4) containing 1 mM CaC12 and 150 mM NaCI] Avidin-alkaline phosphatase or avidin-horseradish peroxidase detection systems (Vector Laboratories, Burlingame, CA; BioMeda, Inc.) Hydrogen peroxide, 30% stock solution (v/v) p-Chloronaphthol (Sigma), 1 mg/ml, in wash buffer containing 20% methanol (v/v) 5-Bromo-4-chloro-3-indoyl phosphate p-toluidine (BCIP; Amresco), 50 mg/ml, in N,N-dimethylformamide Nitroblue tetrazolium chloride (NBT; Amresco), 50 mg/ml, in 50% N,N-dimethylformamide Phosphatase buffer [0.1 M Tris-HCl (pH 9.5) containing 100 mM NaC1 and 50 mM MgCI2] Avidin (Sigma or other source) d-Biotin (Sigma or Pierce) Dialysis membrane (Spectrapor; 6,000 MW cutoff)
Preparation of Sulfo-NHS-Biotin- and NHS-LC-Biotin-Derivatized Calmodulin Purified calmodulin, prepared using melittin-Sepharose, 2~ is dialyzed against coupling buffer, and the concentration is adjusted to 1.0 mg/ml (~.1% ~,280 = 1.8). A 2-rag aliquot of calmodulin solution is adjusted to 1 mM CaCI2 and 0.15 M NaCI. An aliquot (25/.d) of either NHS-LC-biotin (0.66 rag/25/A) or sulfo-NHS- biotin (0.53 mg/25/~1) is added at a 10-fold molar
456
APPLICATIONS
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excess with respect to calmodulin. Previous studies have indicated that the 10:1 molar excess of NHS derivative gives optimal labeling with minimal loss of biological function. 2° The derivatization is carried out for 2 hr at 25 °, and the mixture is then either desalted on a precalibrated Sephadex G-25 column (PD-10, Pharmacia) or dialyzed exhaustively against coupling buffer to remove unreacted reagent. The calmodulin derivatives are stored in coupling buffer with 10% glycerol at - 2 0 °.
Preparation of Photobiotin Acetate-Derivatized Calmodulin Purified calmodulin (in 1 mM CaCI2 and 150 mM NaCI) is dialyzed as described above. In subdued light, photobiotin acetate (0.71 mg/335 ~1) is dissolved in water and added to the calmodulin solution (2 mg total) in a fiat-bottomed tube kept at 4 ° using the precautions and procedures previously outlined. 22 The reaction mixture is then placed 10 cm from a broadspectrum GE sunlamp and irradiated for 10 min at 4°. The reaction mixture is either dialyzed against coupling buffer or chromatographed over a PD-10 Sephadex column to remove unreacted photobiotin. The derivatized calmodulin is stored in coupling buffer containing 10% glycerol at - 2 0 °"
Preparation of p-Diazobenzoylbiocytin-Derivatized Calmodulin p-Diazobenzoylbiocytin (DBB) reagent is activated as previously described23:2 mg of DBB is dissolved in 15/zl of dimethyl sulfoxide. Eightyfive microliters of 1 M HC1 is added, and the mixture is chilled on ice. To this mixture, 40/zl of0.112 M sodium nitrate is added and allowed to react for 5 min. This activation is quenched by adding 100/xl of 1 M NaOH, and the reaction is diluted with 1.75 ml of coupling buffer. In order to start the modification, 1 ml of purified, dialyzed calmodulin (2 mg/ml coupling buffer, brought to 1 m M CaC12and 150 mM NaC1) is reacted with 500/zl of the activated DBB mixture for 1.5 hr at 25°. Following incubation, the reactants are removed by chromatography over PD-10 Sephadex G-25 columns. The derivative is stored in coupling buffer containing 10% glycerol at - 2 0 ° . Characterization of Biotinylated Calmodulin Derivatives A useful method for documenting the production of the various biotinylated species is to determine the ultraviolet spectrum of the calmo22 E. Lacey and W. N. Grant, Anal. Biochem. 163, 151 (1987). 23 M. Wilchek, H. Ben-Hur, and E. A. Bayer, Biochem. Biophys. Res. Commun. 138, 872 (1986).
[53]
457
BIOTINYLATED CALMODULIN DERIVATIVES
dulin before and after derivatization. As can be seen in Fig. 1A-C, all of the modified derivatives result in spectral shifts relative to native calmodulin. Sulfo-NHS-biotin and NHS-LC-biotin derivatives (data not shown) give nearly identical spectra (Fig. 1A), with absorbance maxima shifted from 277 nm (native calmodulin) to 266 nm (NHS-modified calmodulin). Thus, there is no apparent difference in the extent or amount of labeling of calmodulin produced by the more water-soluble sulfo-NHS-biotin reagent or by the more hydrophobic NHS-LC-biotin derivative. Photobiotin (Fig. 1B) and DBB (Fig. 1C) produce a different type of spectral shift, with DBB-modified biotin exhibiting considerable absorbance between 320 and 260 nm. Thus, different chemistries of derivatization result in characteristic spectral patterns of the modified calmodulins. A second method for demonstrating biotinylation is direct visualization of biotinylated calmodulins following transfer to nitrocellulose. Although calmodulin has been reported to bind poorly to nitrocellulose, 24we have found that calmodulin easily and rapidly transfers from 20% polyacrylamide gels to nitrocellulose, provided that the blot is transferred for 1.0
A
U.I 0
Z
.,,,,,.SULFO-BNHS
n." 0
co
NATIVE CaM
0
I
220
I
244
l
I
I
268
I
292
I
~
316
J
340
nm FIG. 1. UV absorbance spectra of biotin-labeled calmodulin preparations. (A) Native calmodulin and NHS-LC-biotin-derivatized calmodulin; (B) photobiotin-modified calmodulin; and (C) DBB-modified calmodulin. All solutions were approximately 2 mg/ml calmodulin in coupling buffer (pH 7.0) and were scanned using a Beckman DU-7 scanning spectrophotometer.
1.0
B
14.1 (..) Z
PHOTOBIOTIN
_~
0
NATIVE CaM
,
'
0 220
244
268
292
316
~40
nm 1.0
C
LU Z
rn nO
NATIV E ~ CaM
0
"'"J
I
220
244
I
"~
L
i
268
I
292 nm
FIG. 1. (continued)
I
,
316
i
340
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
459
only 20 min at 200 mA; normal blotting times of 2-4 hr result in calmodulin passing through the nitrocellulose and into the transfer buffer. In order to further stabilize adsorption of calmodulin on nitrocellulose blots, the freshly transferred blot is immediately fixed in a solution of 10% acetic acid-25% (v/v) 2-propanol for 30 min prior to subsequent processing. Although others have shown that calmodulin will bind to nylon-based membranes, 24 we have found that most nylon membranes result in an unacceptable level of color background when incubated with avidin-based chromogenic enzymes. By reducing the time and current of cell transfer and by fixing the nitrocellulose prior to incubation, satisfactory binding and detection of biotinylated calmodulin on nitrocellulose can be achieved. NHS-LC-Biotinylated calmodulin can be easily detected following transfer to nitrocellulose. For most modifications as little as 20 mg biotinylated calmodulin can be detected. Following dialysis, either 10 or 20 /zg of native or biotinylated calmodulin is subjected to SDS-PAGE (20% gel), transferred, and fixed as described above, and nonspecific binding sites are blocked using blocking buffer for 30 min. Following 3 washes for 15 min each, the blot is incubated with avidin-peroxidase (1 /zg/ml in wash buffer) for 30 min, washed 3 additional times in wash buffer, and reacted with substrate solution, consisting of 0.03% H202 and pchloronaphthol (1 mg/ml) in wash buffer containing 20% methanol. Biotinylated calmodulin is easily visualized following this procedure, while native calmodulin does not generate a signal. Similar results can be obtained with the other calmodulin derivatives. On occasion, particularly if excess molar ratios of derivatization agent are used, several calmodulin peptides of altered electrophoretic mobility can also be visualized on blots; however, these preparations are not effective as probes for blot overlays. 2° The more slowly moving peptides most likely represent calmodulins containing multiple biotinylated amino acid residues and, possibly, having impaired Ca z+ binding, which also decreases electrophoretic mobility. In order to determine whether the biotinylated calmodulin derivatives can recognize calmodulin-binding proteins immobilized on nitrocellulose (Fig. 2), fractions of rat cerebral cortex synaptosomes (lane 1; P2 fraction), cerebral cortical cytoplasm (lane 2; $2 fraction), and partially purified rat cytosolic calmodulin-binding proteins (primarily calcineurin at 60K and caldesmon at 150K and 105K) were subjected to SDS-PAGE and electroblotted. Identical blots are incubated with the biotinylated calmodulin derivatives as follows. Following a 30-min incubation in blocking buffer, blots are washed 3 times for 10 min in wash buffer and 24 C. R. Egly and D. Daviaud, Electrophoresis 6, 325 (1985).
460
APPLICATIONS BNHS
Mr(XJ0 -3) 1
2
PHOTOBIOTIN 3
1
2
3
[53]
DBB 1
2
SULFO-NHS 3
1
2
3
CONTROL 1
2
3
FIG. 2. Relative reactivityof several biotinylatedcalmodulinderivatives. Calmodulin was modifiedwith NHS-LC-biotin(BNHS), sulfo-NHS-biotin,photobiotin, or DBB and incubatedwithblots containingcerebral cortex membranes(lane 1,100v.g),cerebralcortical cytosol (lane 2, 100/~g), or partially purified calmodulin-bindingproteins (lane 3, 10/xg). Followingincubation with biotinylatedcalmodulins,the blots were incubatedwith avidinalkaline phosphatase and developedwith appropriate chromogens.An identicalblot (Control) was reacted with avidin-alkaline phosphatase alone, revealing endogenous biotincontaining peptides having molecularweights of 75,000 and 100,000.
incubated with calmodulin biotinylated using NHS-LC-biotin (BNHS), photobiotin, DBB, or sulfo-NHS-biotin. Each congener is incubated at a concentration of 5/xg/ml in blocking buffer for 30 min, followed by 3 10-min washes in wash buffer. Avidin-alkaline phosphatase complexes (1 /zg/ml wash buffer) are then incubated with the blot for 20 min, followed by 3 additional 10-min washes. A control consists of blotted samples incubated with avidin-alkaline phosphatase alone. The avidin detection system step should not be carried out in the usual blocking buffer because of occasional contamination of the nonfat dry milk with biotin. Each blot is incubated with BCIP-NBT (100 ~1 BCIP stock, 200 t~l NBT stock in 30 ml of phosphatase buffer) until colored bands appear. The color development is carried out under subdued light and can proceed for as long as 12 hr without excessive background staining. As shown in Fig. 2, all biotinylated calmodulin derivatives bind to a number of immobilized calmodulin-binding proteins from rat brain. Some differences in binding were noted, however. NHS-LC-biotin (BNHS) and sulfo-NHS-biotin derivatives of calmodulin recognize several brain
[53]
BIOTINYLATED CALMODULIN DERIVATIVES
461
calmodulin-binding proteins, with prominent staining of peptides at Mr 52,000, 60,000-62,000, 75,000, 85,000, and 105,000-240,000; this pattern is reminiscent of Bio-CaM used in previous studies.14 Control incubations have demonstrated that the peptides at Mr 75,000 and 100,000 bind avidin-alkaline phosphatase alone, suggesting that these peptides contain biotin. DBB-calmodulin gives a less intense pattern of labeling (see staining of the 52,000 peptide) and fails to recognize the 60K subunit of calcineurin (Lane 3). Thus, modification of histidyl and/or tyrosyl residues with DBB appears to alter the recognition of specific binding domains. This may suggest that a specific histidine or tyrosine residue is important for interaction of DBB-calmodulin with immobilized calcineurin. Studies are in progress to investigate this possibility. Additional methods for characterizing the calmodulin derivatives include tests of their ability to stimulate calmodulin-dependent enzymes, limited proteolysis of biotin-calmodulin, and direct sequencing of the modified molecule(s). Past studies have indicated that calmodulin modified with biotin-NHS derivatives retains the ability to stimulate calmodulin-dependent phosphatase activity ~4and calmodulin-dependent phosphodiesterase. 15 Interestingly, Mann and Vanaman observed that although BNHS-calmodulin could stimulate phosphodiesterase, preincubation with avidin inhibited calmodulin stimulation of phosphodiesterase activity. 15 We have observed that phosphodiesterase is poorly detected by BNHS-calmodulin on blot overlays, in agreement with the idea that the biotinylated lysine is obscured following calmodulin interaction with phosphodiesterase. Alternatively, the interaction of avidin may alter the calmodulin conformation such that interaction with phosphodiesterase cannot take place. Mann and Vanaman have sequenced BNHS-calmodulin and found that the biotin label is primarily localized on lysine-94. Thus, biotinylated calmodulin derivatives may prove quite useful in mapping points of interaction between calmodulin and its targets. Biotinylated calmodulin can be purified using a variety of chromatographic techniques. Hydrophobic interaction chromatography can be used to purify calmodulin; however, this method does not resolve biotinylated from native calmodulin. 25 HPLC using DEAE resins has been used to separate modified from native calmodulin. ~5We have used avidinagarose to purify biotinylated calmodulin; however, harsh elution conditions (pH 2.5; 6 M guanidine) must be used (M. Billingsley, unpublished observation). 2s R. Gopalakrishna and W. B. Anderson, Biochem. Biophys. Res. Commun. 104, 830 (1982).
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Uses of Biotinylated Calmodulin
Identification of Tissue-Specific Calmodulin-Binding Proteins The effect of Ca2÷-calmodulin in a given tissue is determined by the expression of particular calmodulin-binding proteins in the various cells of the tissue. We have used BNHS-calmodulin to determine the pattern of calmodulin-binding protein expression in various rat tissues. Membrane preparations, derived from heart, kidney, testis, lung, and pancreas, and equal amounts of tissue protein (100/xg/lane) were subjected to SDS-PAGE and transferred to nitrocellulose. As shown in Fig. 3, each tissue exhibits a distinct pattern of calmodulin-binding proteins. Although some calmodulin-binding proteins are common among tissues, others are specific to a given tissue. Two controls are essential in such studies. One is to determine the Ca 2+ specificity of the biotinylated calmodulin binding by including EGTA in an identical blot incubation (data not shown). Another complementary control is to incubate an identical blot with avidin-alkaline phosphatase alone in order to determine the extent of endogenous biotin-containing proteins in a tissue. It is also possible to block the reactivity of endogenous biotin-containing proteins by incubating the preblocked blot with avidin (0.1 mg/ml in wash buffer containing 1% bovine serum albumin) followed by a wash with excess biotin (1 mg/ml). We have found it easier to locate and "subtract" the biotin containing proteins from the calmodulin-binding proteins, in part because avidin "blocking" can give equivocal results (M. Billingsley and R. Kincaid, unpublished observation).
Detection of Reginal and Subcellular Differences in Calmodulin-Binding Protein Expression Biotinylated calmodulin overlays can also be used to explore the differences in calmodulin-binding proteins in subcellular fractions from brain regions. An example of such a separation is shown in Fig. 4. Rat brain is dissected into cerebral cortex, cerebellum, hippocampus, and striatum. Each tissue is homogenized and separated into synaptosomal (P2) and cytosolic ($2) fractions. Individual lanes of a 10% polyacrylamide gel are loaded with 75/zg total protein, and the resolved proteins are transferred to nitrocellulose. Following blocking as described above, the blot is reacted with NHS-LC-biotinylated calmodulin (5 ~g/ml blocking solution) and detected using avidin-alkaline phosphatase and BCIP-NBT chromagen systems. As shown in Fig. 4, there are marked regional and subcellular differences in calmodulin-binding proteins. Most notable is the relative absence
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463
BIOTINYLATED CALMODULIN DERIVATIVES
PROTEIN STAIN
BIOCAM BLOT
Mr (Xl 0-3)
94--
(57--
43--
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I
30-FIc. 3. Tissue distribution of membrane-associated rat calmodulin-binding proteins. Homogenates of rat heart, kidney, testis, lung, and pancreas were centrifuged at 20,000 g and subjected to S D S - P A G E and blotting as described. The blot was incubated with NHS-LCbiotinylated calmodulin and detected using avidin-alkaline phosphatase systems. The peptide at 75K bound avidin-alkaline phosphatase alone. The 61K marker corresponds to the approximate migration of purified calcineurin.
of the 52K subunit of calmodulin-dependent protein kinase II in cerebellum (lanes 2, 6); this finding has been noted by several investigators. 26,27In addition, there are regional differences in calmodulin-binding peptides between 65K and 87K, with cerebellum exhibiting the greatest difference. The $2 fraction is relatively devoid of high molecular weight calmodulinbinding proteins (i.e., spectrin, caldesmon, and proteolytic fragments thereof), whereas the P2 fraction contains a 36K calmodulin-binding pro26 S. D. Flanagan, B. Yost, and G. Crawford, J. Cell Biol. 94, 743 (1982). 27 T. L. McGuinness, Y. Lai, and P. Greengard, J. Biol. Chem. 260, 1696 (1985).
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Sa rv1 r
1
2
3
4
5
6
7
8
(XlO-3)
94-
67-
43-
36FIG 4. Regional and subcellular distribution of calmodulin-binding proteins in rat cortex
(lanes l, 5), cerebellum (lanes 2, 6), hippocampus (lanes 3, 7), and striatum (lanes 4, 8). Regions were separated into crude synaptosomal (P2) and cytosolic ($2) fractions as described. The blot was incubated with NHS-LC-biotinylated calmodulin and detected using alkaline phosphatase and chromogen systems.
tein termed cytosynalin 28 not seen in the cytosolic fraction. Thus, the biotinylated calmodulin overlay can be used to study regional and subcellular differences in expression of target enzymes. Of note is that, following electrophoresis and blotting, these results are obtained within 3 hr of 28 K. Sobue, T. Okabe, K. Kadowaki, K. Itoh, T. Tanaka, and Y. Fujio, Proc. Natl. Acad. Sci. U.S.A. 84, 1916 (1987).
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BIOTINYLATED CALMODULIN DERIVATIVES
BioCaM
465
Mr (X 10-3) --94 --67
-- 26
-- 18
1
2
3
FIG. 5. Identification of calmodulin-binding peptides after limited proteolysis, SDSPAGE, and transfer to nitrocellulose. Extracts of calmodulin-Sepharose eluates of T lymphocytes (lane 1), splenocytes (lane 2), and thymocytes (lane 3) were incubated for 30 min at 37° with staphylococcal V8 protease, electrophoresed on a 15% polyacrylamide gel in the presence of SDS, and transferred to nitrocellulose. The blot was incubated with biotinylated calmodulin and detected using avidin-alkaline phosphatase systems. T lymphocytes and splenocytes contain a 59K isoform of calcineurin, whereas thymocytes contain a 65K form; digestions indicate nearly identical calmodulin-binding peptides (lanes 2 and 3) from both the 59K and 65K forms. (Adapted, with permission, from Ref. 29.) incubation; similar studies with iodinated calmodulin would take considerably longer.
Proteolytic Mapping of Calmodulin-Binding Peptides W e h a v e a l s o u s e d b i o t i n y l a t e d c a l m o d u l i n o v e r l a y s to o b t a i n " f i n g e r p r i n t s " o f c a l m o d u l i n - b i n d i n g p e p t i d e s f o l l o w i n g l i m i t e d p r o t e o l y t i c di-
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gestion. In a study on the characterization of calmodulin-binding proteins from murine T and B lymphocytes and thymocytes, we have observed that thymocytes contain a major 65,000 calmodulin-binding protein which reacts weakly with antibodies against calcineurin. 29 Since the 59K calmodulin-binding subunit of calcineurin is the major calmodulin-binding peptide in B and mature T lymphocytes, we have used limited proteolysis of both the 65,000 and 59,000 peptides to show substantial relatedness between the calmodulin-binding peptides of these two proteins (Fig. 5). Although this approach may not be applicable to all calmodulin-binding proteins, the results indicate an important potential use of the calmodulin blot-overlay technique. Summary We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulinbinding protein expression, physical points of calmodulin-target interaction, and proteolytic maping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of proteinprotein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, nearphysiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. ~5 One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetyla29 R. L. Kincaid, H. Takayama, M. L. Billingsley, and M. V. Sitkovsky, Nature (London) 330, 176 (1987).
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tion experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. 3° The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins. Acknowledgments This research was supported by a research grant from The InternationalLife Sciences Institute Research Foundation and by U.S. Public Health Service Grant R01-AG06337 to M.L.B. The authors thank Ms. Doris Lineweaverfor manuscriptpreparation. 30 A. S. Manalan and C. B. Klee, Biochemistry 26, 1382 (1987).
[54] A v i d i n - B i o t i n M e d i a t e d I m m u n o a s s a y s : O v e r v i e w
By
M E I R W I L C H E K a n d E D W A R D A . BAYER
Progress in the use of avidin-biotin technology in immunoassays has developed together with major advances of the immunodiagnostics field in general. Thus, whenever a particular improvement in a given step was desired, the applicability of the avidin-biotin complex was rapidly demonstrated. At the beginning, in the late 1960s and early 1970s, when the need for improved diagnostics was established and different approaches (e.g., radioimmunoassay1 and bacteriophage 2 assay systems for the quantification i R. S. Yalow and S. A. Berson, Nature (London) 184, 1648 (1959); D. S. Skelley, L. P. Brown, and P. K. Besch, Clin. Chem. 19, 146 (1973). 20. MS.kel~., Immunology 10, 81 (1966).
METHODS IN ENZYMOLOGY, VOL. 184
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