[58] Structure—Function analyses of mammalian cellular retinol-binding proteins by expression in Escherichia coli

506

ENZYMOLOGY AND METABOLISM

[58]

2

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1-t

o o t!

!

"7, o

0

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I

I

2

4

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all-trans--retinol (I~M)

FIG. 1. Dependence of aU-trans-~ll-cis-retinol isomerization rate on all-trans-retinol concentration. The assays were carried out by incubating 0.30 mg of CHAPS-solubilized protein from nuclear membrane in the presence of 81/zM BSA and variable concentrations of all-trans-retinol in a total volume of 1.1 ml. Data from double reciprocal plot (inset) were used to calculate the apparent Vmaxand Km values for the reaction. described, the rate of 11-cis-retinol formation a p p r o a c h e s saturation at about 5 tzM all-trans-retinol. T h e linear transformation of the saturation c u r v e gives an a p p a r e n t Vmax o f 2.5 n m o l / h r / m g protein and a Km of 1.6 /xM (Fig. 1).

[58] Structure-Function Analyses of Mammalian Cellular Retinol-Binding Proteins by Expression in Escherichia coli By MARC S. LEVIN, ELLEN LI, and JEFFREY I. GORDON Rationale for Prokaryotic Expression of Cellular Retinoid-Binding Proteins Several small ( - 1 5 kDa) intracellular retinoid-binding proteins have b e e n purified f r o m animal tissues and their p r i m a r y structures deterMETHODS IN ENZYMOLOGY, VOL. 189

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

[58]

RAT C R B P AND C R B P II EXPRESSED IN E. coli

507

mined. These include cellular retinol-binding protein (CRBP), 1-3 cellular retinol-binding protein II (CRBP I I ) , 4'5 and cellular retinoic acid-binding protein. 6 They belong to a family of homologous, cytoplasmic, hydrophobic ligand-binding proteins which currently contains 10 known members, several of which bind long-chain fatty acids (reviewed by Sweetser et al.7).

Attempts to rapidly and easily purify these cellular retinoid binding proteins from their cells of origin have been limited by a number of factors. First, the yields of purified protein have been poor: 15/xg of CRBP per gram wet weight of rat liver and 170/zg of CRBP II per gram wet weight of rat small intestine. 8,9 Second, copurification of comparably sized, homologous cellular retinoid-binding proteins may occur when several are expressed in a given tissue. Last, the purified proteins contain bound ligand. To analyze apoprotein-ligand interactions, UV irradiation or organic extraction is required to remove endogenous retinoids, methods which may perhaps lead to structural modifications that could complicate interpretation of subsequent functional studies. By contrast, purification of these mammalian proteins after their expression in Escherichia coli offers several advantages: (1) large quantities of protein are easily obtained; (2) only one retinoid-binding protein is expressed because E. coli does not possess any endogenous retinoid-binding proteins; (3) because E. coli does not require retinoids for growth and endogenous retinoids are not present, the retinoid-binding proteins purified from this prokaryote can be obtained as apoproteins; (4) site-directed mutagenesis of cloned cDNAs encoding retinoid-binding proteins and their subsequent expression in E. coli represent a powerful method for analyzing protein structure-activity relationships; and (5) amino acid derivatives can be introduced into the proteins by growing suitable E. coli auxotrophs containing a prokaryotic expression vector in the presence of the analog. Analog-substituted retinoid-binding proteins can, in turn, be used for nui M. M. Bashor, D. O. Tort, and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 70, 3483 (1973). 2 D. E. Ong and F. Chytil, J. Biol. Chem. 253, 828 (1978). 3 j. Sundelin, H. Anundi, L. Tragardh, U. Erikson, P. Lind, H. Ronne, P. A, Peterson, and L. Rusk, J. Biol. Chem. 260, 6488 (1985). 4 D. E. Ong, J. Biol. Chem. 259, 1476 (1984). 5 E. Li, L. A. Detainer, D. A. Sweetser, D. E. Ong, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 83, 5779 (1986). 6 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6495 (1985). 7 D. A. Sweetser, R. O. Heuckeroth, and J. I. Gordon, Annu. Reo. Nutr. 7, 337 (1987). 8 D. E. Ong, A. J. Crow, and F. Chytii, J. Biol. Chem. 257, 13385 (1982). 9 W. H. Schaefer, B. Kakkad, J. A. Crow, I. A. Blair, and D. E. Ong, J. Biol. Chem. 264, 4212 (1989).

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ENZYMOLOGY AND METABOLISM

[58]

clear magnetic resonance (NMR) studies of ligand-protein interactions (see below). Expression of Cellular Retinol-Binding Proteins in Escherichia coli General Principles For a general discussion of useful strategies for expressing, identifying, and characterizing recombinant gene products in E. coli, the reader is referred to the review by Shatzman and Rosenberg in this series.l° Two desirable components of prokaryotic expression vectors are (I) inducible promoters which can direct efficient transcription of foreign cDNAs and (2) translational control elements that allow efficient initiation of translation of the foreign mRNA transcript to occur. We have used pMON vectors 11 designed and provided by Monsanto z but a large number of commercially available expression vectors are also suitable. 12The pMON series of vectors are derived from pBR32713 and incorporate the E. coli recA promoter which can be induced by nalidixic acid and a ribosome binding site from the T7 bacteriophage gene 10 leader (G10L) sequence. II The pMON vectors that we have used contain a unique NdeI restriction site downstream from the recA promoter and the translational control element. By genetically engineering an NdeI site at its initiator ATG codon (if necessary), the cDNA of interest can be placed under the control of the recA promoter. Moreover, the distance between the vectorderived G10L ribosome-binding site and its translation start site is thereby maintained. One of the advantages of such a vector is that it directs the synthesis of a full-length "recombinant" polypeptide rather than one which represents a fusion between bacterial and eukaryotic protein sequences. The bacterial strain used for expression of the recombinant protein must be selected with several caveats in mind. First, the induction system used must be compatible with the bacterial phenotype. For example, a recA- strain cannot be used if the E. coli recA promoter is utilized. Second, E. coli proteases such as the product of the lon gene (protease La) may cause proteolysis of some foreign proteins. Expression of these proteins may be improved by using strains that are protease deficient (e.g., Lon- and Htpr-). Third, variables that can affect the efficiency of ~0A. R. Shatzman and M. Rosenberg, this series, Vol. 152, p. 661. H p. O. Olins, C. S. Devine, S. H. Rangwala, and K. S. Kavka, Gene 73, 227 (1988). lz Examples of currently available vectors that are suitable for production of "nonfusion" proteins in E. coli include pBTac2 and pBTrp2 (Boehringer Mannheim Biochemicals); pNHI8A (Stratagene); and pKK233-2 and pPL Lambda (Pharmacia LKB Biotechnology). 13 X. Sober6n, L. Covarrubias, and F. Bolivar, Gene 9, 287 (1980).

[58]

RAT CRBP AND CRBP II EXPRESSEDIN E. coli

509

production of recombinant proteins include incubation temperature, growth medium, cell density, timing of induction, and the length of fermentation after induction of foreign protein synthesis. Finally, it should also be noted that E. coli cannot support many critical posttranslational modifications of mammalian proteins (e.g., glycosylation). Growth o f Escherichia coli Transformed with p M O N - C R B P or pMON-CRBP H

We have not found it necessary to utilize protease-deficient hosts for the expression of CRBP and CRBP II. For most purposes, we use E. coli strain JM101 transformed with pMON-based vectors (Monsanto). A fresh overnight culture~ of E. coli JM101 containing the recombinant pMON vector is diluted 1 : 50 in fresh Luria broth plus ampicillin (100 /~g/ml) (pMON contains an A m p r locus). After achieving a cell density equivalent to an OD600 of approximately 0.2 nm, nalidixic acid is added (1:200 dilution of a 10 mg/ml stock prepared in 0.1 N NaOH) to activate the recA promoter of the plasmid. Fermentation is continued with agitation for an additional 3 hr at 30° (at which time the OD60o is -1.0). Cells are then harvested by centrifugation at 5,000 g or by filtration through a Millipore (Bedford, MA) Pellicon cassette (the Pellicon cassette is useful for harvesting cells from large-scale cultures, i.e., 20-100 liters). The cell paste is stored at - 7 0 °. Purification o f Escherichia coli Expressed Rat CRBP and CRBP H

Although foreign proteins that are expressed in E. coli may be soluble, many are present as insoluble aggregates known as inclusion bodies (reviewed by Marstonl4). Recovery of active proteins from these aggregates requires isolation of the inclusion bodies following cell lysis, subsequent denaturation to solubilize the protein, and finally a refolding reaction. Because soluble proteins can be purified directly, they are more likely to retain their native conformation. At the conclusion of the fermentation described in the preceding section, rat CRBP or CRBP II represents 1015% of the total soluble proteins present in E. coli homogenates. The purification of rat apo CRBP and apo CRBP II from E. coli is remarkably straightforward and relatively rapid, requiring two or t h r e e steps. 15,16Our protocol is summarized in Table I. An aliquot of the E. coli i4 A. Marston, Biochem. J. 240, 1 (1986). 15 E. Li, B. Locke, N. C. Yang, D. E. Ong, and J. I. Gordon, J. Biol. Chem. 262, 13773 (1987). t6 M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, J. Biol. Chem. 263, 17715 (1988).

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ENZYMOLOGY AND METABOLISM

[58]

TABLE I PURIFICATIONOF RAT APo-CRBP AND APo-CRBP II FROMEscherichia coli LYSATES 1. 2. 3. 4.

5. 6. 7. 8. 9.

Thaw cell pellet and resuspend in 2-3 volumes cold lysis buffer (see text). Disrupt bacteria with a French press (-2000 psi). Clear lysate by centrifugation at 5000 g for 10 min (4°). Ammonium sulfate fractionation: Drip in an equal volume of cold filtered 100% saturated ammonium sulfate (Whatman) containing 15 mM dithiothreitol (pH adjusted to 7.1 with KOH) over 4 hr (keep on ice). Centrifuge as above to obtain a 50% ammonium sulfate supernatant. For CRBP II only, bring the 50% supernatant to 70% saturation by the addition of solid ammonium sulfate. Centrifuge as above to obtain the 50-70% pellet. Suspend this pellet in lysis buffer. Dialyze the ammonium sulfate fractions (-0.1-0.2 liters) overnight at 4° against -20 liters potassium phosphate (20 mM, pH 7.1), EDTA (1 mM), 2-mercaptoethanol (10 mM), sodium azide (0.05%), phenylmethylsulfonyl fluoride (0.02%, pH 7.1). Concentrate the postdialysis preparations, if necessary, to afinal protein concentration of - 5 mg/ml with an Amicon YMI0 membrane. Perform gel-filtration column chromatography with Sephadex G-50. Screen fractions by absorbance at 280 nm and by SDS-PAGE. Pool fractions containing CRBP or CRBP II. If the acyl cartier protein of E. coli is present (detected by SDS-PAGE and aminoterminal amino acid sequencing) or if separation of Met + from Met- CRBP is desired, then proceed to FPLC, Step 9. Perform FPLC with a Mono Q (Pharmacia) column equilibrated with imidazole (20 mM, pH 6.8) and eluted with a NaC1 gradient (see Fig. IB for details).

cell paste is first thawed to r o o m t e m p e r a t u r e and resuspended in 2 - 3 volumes of lysis buffer [Tris (final concentration 50 m M , p H 7.9), sucrose (10%), and p h e n y l m e t h a n e s u l f o n y l fluoride (0.5 mM)]. Bacteria are disrupted b y p a s s a g e through a F r e n c h Press ( - 2 0 0 0 psi). Cellular debris is r e m o v e d b y centrifugation at 5000 g for 10 min at 4 °. A m m o n i u m sulfate fractionation of the supernatant fraction is a v e r y useful first step in the purification: m o s t bacterial proteins are insoluble in solutions of NH4SO4 at 50% saturation, in contrast to the cellular retinol-binding proteins which remain soluble. Thus, the supernatant is adjusted slowly to 50% saturation with NH4SO4 (with constant stirring at 0-4°). After gentle stirring for an additional hour, the suspension is subjected to centrifugation at 41,000 g for 30 min at 4 °. An additional 70% NH4SO4 " c u t " is also beneficial w h e n purifying C R B P II. The o v e r 50% NH4SO4 supernatant containing C R B P or the 50-70% NH4SO4 precipitate containing C R B P II is then dialyzed overnight at 4 ° against buffer A [potassium p h o s p h a t e (20 m M , p H 7.1), E D T A (1 m M ) , glycerol (15%), sodium azide (0.05%), phenylmethanesulfonyl fluoride (0.5 m M ) , and 2 - m e r c a p t o e t h a n o l (10 mM)]. The dialyzed protein solution is then fractionated through a S e p h a d e x G-50-80 column equilibrated with the s a m e buffer. C o l u m n fractions are a s s a y e d for protein b y mea-

[58]

RAT C R B P AND C R B P II EXPRESSED IN E. coil

511

suring the absorbance at 280 nm. Selected fractions are then surveyed by electrophoresis through a 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS-PAGE). Typically, CRBP and CRBP II elute from the gel-filtration column at a relative retention volume (Ve/Vo) of 1.5 (see Fig. 1A). When purifying CRBP (but not CRBP II) an additional purification step is desirable to avoid contamination with the acyl carrier protein of E. coli. The acyl carrier protein is a soluble 8.8-kDa protein which migrates on SDS-PAGE as an approximately 20-kDa protein and elutes like a 12-kDa protein from Sephadex G-50 (reviewed by Rock and Cronan17). Those fractions which appear to be enriched for CRBP by SDS-PAGE are pooled and further purified by fast protein liquid chromatography (FPLC). The pooled fractions are applied to a Pharmacia (Piscataway, NJ) Mono Q column equilibrated with imidazole (20 mM, pH 6.8) and sodium azide (0.05%). CRBP is eluted with a continuous NaCI gradient (15.5-25 mM) in the same buffer. Two CRBP peaks elute at approximately 19 and 20 m M NaC1 (see Fig. 1B). These peaks correspond to CRBP without and with its initiator methionine residue, respectively. The proportion of Met + CRBP and Met- CRBP varies from protein preparation to preparation. The initiator methionine of foreign proteins expressed in E. coli may be removed coincidently, or shortly after synthesis, by a nonspecific aminopeptidase. ~8,19 The efficiency of methionine removal from recombinant proteins is variable and affected by the physicochemical properties of the penultimate amino acid. 18,~9The spectrofluorimetric and ligand binding properties of Met + and Met- CRBP appear to be identical. 16 For some applications, such as protein crystallization and NMR studies, it may be desirable to separate the two species (see below). Protein integrity and purity can be assessed by SDS-PAGE, isoelectric focusing, and automated sequential Edman degradation. Unlike the forms recovered from mammalian cells or cell-free translation systems (wheat germ or reticulocyte lysates), E. coli-derived rat CRBP and CRBP II do not have an acetyl group linked to their amino-terminal amino acid. 15,16Therefore, the intact E. coli-derived proteins can be sequenced directly. Purified E. coli-derived rat apo-CRBP and apo-CRBP II are stored in plastic tubes at 4° in phosphate buffer (20 mM, pH 7.4) supplemented with 2-mercaptoethanol (1 mM), EDTA (1 mM), and NaN3 (0.05%). We have not found aggregation to be a problem even at protein concentrations as high as 15 mg/ml ( - 1 mM). 17 C. O. Rock and J. E. Cronan, Jr., this series, Vol. 71, p. 341. 18 j. L. Brown, Biochim. Biophys. Acta 221, 480 (1970). 19j. L. Brown and J. F. Krall, Biochem. Biophys. Res. Commun. 42, 390 (1971).

512

ENZYMOLOGY AND METABOLISM

[58]

A vo 66453629-

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FIG. 1. Purification o f rat C R B P from E. coli lysates. (A) Fractionation of a m m o n i u m sulfate fractions by S e p h a d e x G-50 c h r o m a t o g r a p h y . A 5 × 150 c m Sephadex G-50 c o l u m n was pretreated with 11 m g o f bovine s e r u m albumin (dissolved in 25 ml of buffer A, described in the text) and t h e n equilibrated with several c o l u m n v o l u m e s o f buffer A. Approxi-

[58]

RAT CRBP AND CRBP II EXPRESSEDIN E. coli

513

Quantitation A reliable measure of CRBP and CRBP II concentration is required for many experiments (e.g., determination of ligand binding constants and stoichiometries). We have determined that the molar extinction coefficients for E. coli-derived CRBP and CRBP II are 28,080 and 25,506 M-~ cm -~ , respectively. 15,16The concentrations of solutions of purified CRBP and CRBP II determined using these values are within 5% of those derived using a protein assay kit (Bio-Rad, Richmond, CA) based on the procedure of Bradford 2° with bovine serum albumin as a standard. Conformational Analysis The spectrofluorimetric properties of rat CRBP and CRBP II purified from liver/intestine have been well characterized. 2,4 Our observation that purified E. coil-derived CRBP and CRBP II do not absorb above 310 nm confirms that they do not contain any endogenous (bound) retinol2 5,~6 Moreover, when excitation and emission spectra are collected from E. coli-derived CRBP and CRBP II complexed with a molar excess of alltrans-retinol, the results are virtually identical to those obtained from the respective rat liver and intestinal holoproteins (see Fig. 2). These latter observations suggest that purified E. coli-derived rat holo-CRBP and holo-CRBP II have folded into a (overall) conformation which is similar to that of the "authentic" tissue proteins. 2o M. M. Bradford, Anal. Biochem. 72, 248 (1976).

mately 100 ml of the postdialysis 50% NH4SO4fraction containing around 360 mg protein was subsequently applied. Fractions (20 ml) were collected at 4° (flow rate 20 mi/hr). The OD280 readings of selected fractions are shown plotted against the total elution volume. Twenty-five microliters of selected Sephadex G-50 fractions were reduced, denatured, and then fractionated by electrophoresis through a 15% polyacrylamide gel containing sodium dodecyl sulfate (0.1%). The results obtained after Coomassie blue staining of this gel are shown in the inset. The elution volume of each of the fractions is labeled. The position of migration of authentic rat liver CRBP is noted by the arrow. Mr markers are indicated at left. (B) Separation of Met + and Met- CRBP by fast protein liquid anion-exchange chromatography. Sephadex G-50 fractions containing CRBP were pooled. They were then equilibrated with imidazole buffer (20 mM, pH 6.8, plus 0.05% sodium azide) and concentrated using a Centriprep 10 concentrator (Amicon). Six milliliters of this protein solution ( - 5 mg) was loaded onto a 5 m m x 5 cm Mono Q HR 5/5 column (Pharmacia). The protein solution was fractionated using the NaCI gradient shown (flow rate 1 ml/min; temperature 22-25°). The OD2s0 was monitored continuously.

514

ENZYMOLOGY AND METABOLISM

A

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I

250

I

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300 350 WAVELENGTH,nm

FIG. 2. Comparison of the spectral properties of E. coli-derived rat CRBP and CRBP II complexed with all-trans-retinol with the corresponding authentic tissue-derived proteins. Approximately 1.5 to 1.8 heM solutions of CRBP or CRBP II were used, and all measurements were made at 25°. Escherichia coli-derived CRBP or CRBP II are represented by thin lines, whereas rat liver CRBP or rat intestinal CRBP II are denoted by thick lines. (A, C) Absorption spectra of CRBP and CRBP II, respectively, complexed with all-trans-retinol. These spectra were taken on a Perkin-Elmer Lambda 5 UV-Vis spectrophotometer (slit width 2 rim). (B, D) Corrected fluorescence excitation and emission spectra of CRBP and CRBP II, respectively, complexed with all-trans-retinol. These spectra were obtained using a Perkin-Elmer MPF-66 spectrofluorimeter equipped with corrected spectra units and constant temperature cell holders.

Ligand-Protein Interactions S p e c t r o f l u o r i m e t r i c m e t h o d s d e s c r i b e d b y C o g a n et al. 21 c a n b e u s e d to c o m p a r e t h e affinities a n d b i n d i n g s t o i c h i o m e t r i e s o f E . c o l i - d e r i v e d a p o - C R B P a n d a p o - C R B P II for a v a r i e t y o f l i g a n d s (see Refs. 15, 16, a n d 21 U. Cogan, M. Kopelman, S. Mokady, and M. Shinitzky, Eur. J. Biochem. 65, 71 (1976).

I

400

RAT C R B P AND C R B P II EXPRESSED IN E. coli

[58]

515

TABLE II PHYSICOCHEMICAL PROPERTIES OF Escherichia

coli-DErOVED RAT CRBP AND CRBP II Property pl E2~0 (M -1 cm -I) Fluorescence (maxima) Excitation (nm) c Emission (nm) d Stoichiometry of ligand binding all-trans-Retinol binding (K~, nM) all-trans-Retinal binding (Kr, nM) all-trans-Retinal competes with all-trans-retinol for binding all-trans-Retinoic acid binding Methylretinoate binding

CRBP a

CRBP IP .b

4.73 4.78 28,080

5.61 5.66 25,506

282 338 1: 1 20 50 No

282 336

No No

1:1

10 90 Yes No No

a M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, J. Biol. Chem. 263, 17715 (1988). b E. Li, B. Locke, N. C. Yang, D. E. Ong, and J. I. Gordon, J. Biol. Chem. 262, 13773 (1987). c Emission: h = 340 nm. d Excitation: h = 290 nm.

Table II). When all-trans-retinol is bound to CRBP or CRBP II, it has a distinct absorption spectra and exhibits enhanced fluorescence emission compared with that of unbound all-trans-retinol. 2,4 Quantitative binding studies can be done by exploiting these differences. Apo-CRBP and apoCRBP II both contain four tryptophan residues in comparable positions (9, 89, 107, and 110). When the apoproteins are excited at around 290 nm, they demonstrate fluorescence with an emission maxima near 340 nm. However, when saturated with all-trans-retinol, 90% of the intrinsic protein (tryptophan) fluorescence is quenched. Because the efficiency of energy transfer is dependent on the distance between the tryptophan residue(s) and the bound retinol (otr-6), this would suggest that at least one tryptophan residue in each of these proteins is located near the binding site. The capacity of nonfluorescent substances to bind to CRBP and CRBP II can also be assessed by monitoring their ability to quench the native fluorescence of these proteins. Ligand binding studies have shown that both apo-CRBP and apoCRBP II bind all-trans-retinal as well as all-trans-retinol with high affinity ( T a b l e 11). 16 Neither can bind all-trans-retinoic acid nor methyl retinoate,

516

ENZYMOLOGY AND METABOLISM

[58]

an uncharged analog of retinoic acid.16 The latter observation suggests that the inability to bind all-trans-retinoic acid is not simply due to the negative charge of the C-15 carboxylate group. Although both proteins can bind all-trans-retinal with high affinity in direct binding assays, monitoring of retinol fluorescence revealed that all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. 16,22These observations raised the possibility that CRBP and CRBP II do not complex to alltrans-retinal in the same way, despite the fact that their apparent Kd values are essentially identical (Ref. 16 and below). Isotopic Labeling of Retinoid-Binding Proteins Expressed in Escherichia coli for Nuclear Magnetic Resonance Analysis NMR spectroscopy provides information about the structural environment, chemical properties, and motional characteristics of defined functional groups in protein molecules. Detection of site-specific changes in chemical shifts in the NMR spectra can be used to follow protein conformational changes during processes such as ligand binding, protein-lipid interactions, and denaturation-renaturation. Although considerable information about protein structure and dynamics has been obtained by the use of IH NMR, assignment of resonances is a formidable task in the size range of the intracellular retinoidbinding proteins ( - 1 5 kDa). Isotopic labeling of a particular amino acid with a variety of magnetic nuclei (EH, ~3C, ~5N, and ~gF) has been used to simplify the NMR spectrum ("spectral editing") of a number of proteins. 23 Incorporation of isotopically labeled amino acid analogs can be facilitated by selecting strains of E. coil which are auxotrophs for the amino acid of interest and using them to express foreign proteins. 19F nuclei are very useful as NMR probes. Their advantages include 100% natural abundance, a sensitivity close to that of protons, a large chemical shift range, and the absence of background signals. ~9F NMR spectroscopy represents an alternative to fluorescence spectroscopy for studying CRBP/CRBP II-retinoid interactions. Escherichia coli provide a straightforward way of obtaining large quantities of the 19F-labeled cellular retinoid-binding proteins. To do so, a tryptophan auxotroph (E. coli strain w3110 trp A 3 3 ) 24 is transformed with the recombinant pMON expression vector containing CRBP or CRBP II cDNA. An overnight culture of transformed cells is diluted 1 : 15 in M9 medium 25 supplemented with 0.25% glucose, I% casamino acids, 0.1% 22 p. N. MacDonald and D. E. Ong, J. Biol. Chem. 262, 10550 (1987). 23 j. L. Markey and E. L. Ulrich, Annu. Rev. Biophys. Bioeng. 13, 493 (1984). 24 G. R. Drapeau, W. J. Brammer, and C. Yanofsky, J. Mol. Biol. 35, 357 (1968).

[58]

RAT CRBP AND CRBP II EXPRESSEDIN E. coil

517

vitamin Bl, 5 mg/liter FeCl3, 0.4 mg/liter ZnSO4, 0.7 mg/liter CoCl2, 0.7 mg/liter Na2MoO4, and 0.1 mM L-tryptophan and incubated at 30°. After achieving an OD600 of 1.0, cells are harvested by centrifugation at 5000 g and resuspended in the above medium except that 6-fluorotryptophan (final concentration 0.1 mM) is substituted for L-tryptophan. The cells are incubated at 37° for 30 min, and expression of the cellular retinol-binding protein is induced by adding nalidixic acid to a final concentration of 50/xg/ml. Following a 3-hr fermentation, cells are harvested, and the 6-fluorotryptophan-substituted rat cellular retinol-binding protein purified as detailed above. The purified protein solution is adjusted to 1 mM by ultrafiltration through YM10 filters (Amicon, Lexington, MA) and then equilibrated with D20 by four cycles of buffer exchange using Centriprep 10 concentrators (Amicon). The efficiency of incorporation of fluorotryptophan analogs into CRBP or CRBP II is high, greater than 90%. This value was obtained by comparing the NMR signal intensities of known amounts of the fluorotryptophan-labeled protein and trifluoroacetic acid (the latter is included in the sample as an internal reference standard. The presence of analog does not alter the ligand binding properties of the protein as measured by fluorescence spectroscopic methods. The choice of amino acid analog (for example 4-fluoro-, 5-fluoro-, or 6fluorotryptophan) is empirical and based on the resolution of spectral signals obtained from individual groups with a given analog. With CRBP II, a 1.5-liter culture yields sufficient amounts of purified 6-fluorotryptophan-substituted protein (5-10 mg) to obtain high-quality spectra (0.8 ml of a 1 mM solution). At this protein concentration, using a Varian VXR500 spectrometer, as few as 200-300 transients can be collected to obtain spectra with an excellent signal-to-noise ratio. Spectra can be obtained (with a larger number of transients) using solutions that are 10-fold more dilute. Figure 3 shows uncoupled 470.3-MHz 19F NMR spectra of a 1 mM solution of 6-fluorotryptophan-substituted E. col#derived rat CRBP II in the absence of ligand and fully saturated with all-trans-retinol. Resonances (labeled A and B) corresponding to two of the tyrptophan residues (WA and WB), undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, the resonances labeled C and D, corresponding to two other tryptophan residues (Wc and WD), undergo only minor perturbations in chemical shifts. The fact that only two of the four resonances arising from the four 25 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

518

ENZYMOLOGY AND METABOLISM

[58]

AB

a Apo CRBP II ~l

C

D

t

b

/

D A

-

42

C

- 44

-46

-48

-

50

PPM FROM TFA

FIG. 3. 19F NMR spectra (recorded at 470.3 MHz) of 6-fluorotryptopban-labeled E. coliderived rat CRBP II with and without bound all-trans-retinol. (a) Spectrum collected on a Varian VXR 500 spectrometer using a 1 m M solution of the apoprotein at 22° (1344 transients). (b) Spectrum obtained at 22° with a 1 m M solution of protein after incubation with 1.1 m M all-trans-retinol (896 transients). A line broadening of 4 Hz was applied. The chemical shifts are referenced to the 19F signal for trifluoroacetic acid.

tryptophan residues are sensitive to the binding of all-trans-retinol provides a functional assay for (1) examining the conformational effects of the binding of a series of retinoids with different structures to a given cellular retinoid-binding protein and (2) comparing the effects of binding a given retinoid on the structures of several retinoid-binding proteins. For example, comparison of the ]9F NMR spectra of 6-fluorotryptophan-substituted CRBP and CRBP II with and without bound all-trans-retinol and all-trans-retinaldehyde can provide an independent evaluation of the fluorescence spectroscopic data alluded to above, which suggested that alltrans-retinaldehyde interacts with each protein in a unique way. It is important to note that the ]9F resonances can be extremely sensitive to amino-terminal heterogeneity in the protein sequence. The relative intensities of the two signals comprising resonances B, C, D in Fig. 3 correspond to the relative amounts of two protein species: Met + CRBP II, which has the initiator methionine, and Met- CRBP II, with an amino-

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terminal Thr residue (the second amino acid residue in the primary translation product). Thus, because of the sensitivity of 19F NMR, care must be taken to purify Met + from Met- CRBP II. T h e 19F NMR spectra will be much easier to interpret once each resonance (WA--WD) can be definitively assigned to each of the four tryptophan residues in the cellular retinol-binding protein and when the tertiary structure of these proteins are known. The assignments may be facilitated by systematic site-directed mutagenesis of CRBP/CRBP II cDNA so that conservative substitutions are made for each Trp residue (e.g., replacement with another aromatic amino acid such as Phe). A comparison of the 19F NMR spectra of the 6-fluorotryptophan-labeled wild-type and mutant proteins could then be undertaken. Resonances corresponding to the substituted tryptophan residue would be predicted to be absent in the 19F NMR spectra of the mutant protein. Crystallization of Cellular Retinol-Binding Proteins Both bovine liver CRBP 26 and E. coli-derived CRBP I127 have been crystallized, although the tertiary structures have not yet been solved. The conditions used to crystallize the E. coil-derived protein without bound retinol are remarkably similar to those used to crystallize the homologous CRBP. A 10 mg/ml solution of protein is prepared in a buffer containing 30 mM Tris, 75 m M NaCI, 2 mM EDTA, 2 mM dithiothreitol, and 0.05% sodium azide (final pH 8.0). Six microliters of this CRBP II preparation is mixed with an equal volume of a buffer containing 37% polyethylene glycol 4000 (Baker), 2 mM cadmium acetate, 2 mM EDTA, 2 m M dithiothreitol, and 0.05% sodium azide (final pH 7.9). Mixing takes place on a dimethyldichlorosilane-treated glass coverslip. The drop is then inverted over a diffusion chamber containing the polyethylenecontaining buffer. Crystals usually appear within 1-2 weeks when using the hanging drop vapor diffusion method. The CRBP II crystals are triclinic with a Pl space group. The unit cell contains two monomeric copies of the 134-residue protein. Its dimensions are as follows: a = 36.8 A, b = 64.0/~, c = 30.4 ~, a = 92.8 °,/3 = 113.5°, y = 90.1 °. Solution of the structures of crystalline CRBP and CRBP II should be aided by the recent determination of the structures of two homologous intracellular hydrophobic ligand binding proteins, namely, rat intestinal fatty acid-binding 26 M. E. Newcomer, A. Liljas, U. Erikkson, L. Rask, and P. A. Peterson, J. Biol. Chem. 256, 8162 (1981). 27 j. C. Sacchettini, D. Stockhausen, E. Li, L. J. Banaszak, and J. I. Gordon, J. Biol. Chem. 262, 15756 (1987).

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protein 28 and the P2 protein from bovine peripheral nerve, z9 Comparison of the structures of apo-CRBP II/CRBP with that of the corresponding holoproteins should provide important insights about the mechanisms of ligand binding and the effects of the bound retinoid on protein structure. Acknowledgments Work from our laboratories cited in this chapter was supported by grants from National Institutes of Health (DK 30292), the Lucille P. Markey Charitable Trust Foundation, and the Monsanto Company. E.L. is a Lucille P. Markey Scholar. J.I.G. is an Established Investigator of the American Heart Association. We would like to acknowledge the contributions of Nien-chu C. Yang and Bruce Locke (University of Chicago), David Ong (Vanderbilt University), James C. Sacchettini, Andre d'Avignon, and Shi-jun Qian (Washington University), plus Leonard J. Banaszak (University of Minnesota) and Peter Olins (Monsanto) to various aspects of these studies. 2s j. C. Sacchettini, J. I. Gordon, and L. J. Banaszak, J. Mol. Biol. 208, 327 (1989). 29 T. A. Jones, T. Bergfors, J. E. Sedzik, and T. Unge, EMBO J. 7, 1597 (1988).

[59] N A D ÷ - D e p e n d e n t R e t i n o l D e h y d r o g e n a s e Liver Microsomes

in

By M. A. LEO and C. S. LIEBER Introduction The classic pathway for the conversion of retinol to retinal in the liver involves a cytosolic NAD÷-dependent retinol dehydrogenase (CRD), believed to be similar, if not identical, to liver alcohol dehydrogenase (ADH, alcohol : NAD + oxidoreductase, EC 1.1.1.1). l,z Our previous observation that a strain of deermice lacks this enzyme without apparent adverse effects 3 prompted a search for an alternate pathway for the production of retinal, the precursor of retinoic acid. Evidence was obtained in favor of the existence of an NAD+-dependent microsomal retinol dehydrogenase (MRD) 4 which can convert retinol to retinal using NAD + as a cofactor. It is distinct from the cytochrome P-450 microsomal system on the one

R. D. Zachman and J. A. Olson, J. Biol. Chem. 236, 2309 (1961). 2 E. Mezey and P. R. Holt, Exp. Mol. Pathol. 15, 148 (1971). 3 M. A. Leo and C. S. Lieber, J. Clin. Invest. 73, 593 (1984). 4 M. A. Leo, C. I. Kim, and C. S. Lieber, Arch. Biochem. Biophys. 259, 241 (1987).

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