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Formation of intraprotamine disulfides in vitro
ARCHIVESOFBIOCHEMISTRYANDBIOPHYSICS Vol.
296, No. 2, August
1, pp. 384-393,
1992
Formation of lntraprotamine
Disulfides in vitro’
Rod Balhorn,’ Michele Corzett, and J. A. Mazrimas Biomedical
Received
Division,
July
L-452, Lawrence
1, 1991, and in revised
form
Livermore
March
National
Laboratory,
Livermore,
California
94550
20, 1992
When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solublization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein’s cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the aminoand carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro. Q 1992 Academic PWS, 1110.
Protamines are small, highly charged proteins that package the DNA of most vertebrate sperm in a highly
condensed, genetically inactive state. While the elucidation of the primary sequences of several fish protamines (l-11) has revealed that these proteins are little more than polyarginine molecules interspersed with occasional uncharged or phosphorylatable amino acids, the protamines of mammals (11-21) are considerably more complex. Two very different protamines have been isolated from mammalian sperm. Protamine 1, the smaller of the two proteins, contains 50 amino acids and is nearly twice the size of many fish protamines. The second molecule, protamine 2, has been isolated from a variety of species (22) but only the proteins from mouse (23), human (11, 18, 24-26), stallion (27), and hamster (19, 28) sperm have been sequenced. These proteins are larger than protamine 1 and each has a composition and primary sequence that differs dramatically from the known sequences of protamine 1. Primary sequences have been determined for protamine 1 isolated from bull (12, 17), ram (14), boar (13), mouse (Xi), hamster (19), and human (16, 18, 26) sperm. Comparisons of these protein sequences have demonstrated that the majority of the amino acid sequence in each molecule is highly conserved. The amino-terminus of each protein begins with the same sequence, ala-arg-tyr-argcys-cys. The central half of protamine contains tandemly repeated polyarginine stretches and resembles the entire molecule of fish protamine, both in sequence and size. Unlike the protamines of fish, however, mammalian protamines contain cysteine residues that are intercrosslinked, locking adjacent protamines around the DNA molecule (29-33). These proteins also contain carboxyl- and aminoterminal peptide sequences that are relatively deficient in arginine and appear to interact with other protamines or sperm nuclear proteins.
1 This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any States Government or the University of California. The views and opininformation, apparatus, product, or process disclosed, or represents that ions of authors expressed herein do not necessarily state or reflect those its use would not infringe privately owned rights. Reference herein to of the United States Government or the University of California, and any specific commercial products, process, or service by trade name, shall not be used for advertising or product endorsement purposes. trademark, manufacturer, or otherwise, does not necessarily constitute ’ To whom correspondence should be addressed. or imply its endorsement, recommendation, or favoring by the United 384 All
Copyright 0 1992 rights of reproduction
0003-9861/92 $5.00 by Academic Press, Inc. in any form reserved.
FORMATION
OF
INTRAPROTAMINE
Studies in this laboratory have revealed that the aminoand carboxyl-terminal ends of each bull protamine bound to DNA in mature sperm must be folded in toward the center of the molecule and locked in place by a disulfide bond (34). During maturation of the sperm in the epididymis, over half of the cysteine residues in the molecule are crosslinked through intramolecular disulfide bridges (34). We describe studies with bull protamine which demonstrate that this protein rapidly forms disulfide-stabilized folded structures in solution and that these structures are similar to those isolated from synthetic DNAprotamine complexes and partially reduced bull sperm. MATERIALS
AND METHODS
Isokzttin and purification of bull protamine. Bull semen was purchased from the American Breeders Service, DeForest, Wisconsin. Boar semen was obtained from Swine Genetics International, Inc., Cambridge, Iowa. Bull, boar, and mouse sperm basic proteins were isolated from frozen semen or epididymal sperm (mouse) as previously described (35). Protamine was separated from the mixture and purified by HPLC on a Nucleosil RP-Cl8 column using the ion-pairing agent trifluoroacetic acid (TFA)3 and a linear acetonitrile gradient (17). The fractions containing bull protamine were collected and lyophilized. Gel electrophoresis. Reduced, modified, and folded protamine samples were analyzed by disc gel electrophoresis using lo-cm acid-urea gels (35,36). These 15% acrylamide gels were preparedwith 0.4% crosslmking and contained 2.5 M urea. Samples were dissolved in minimal volumes of 0.9 N acetic acid, 20% sucrose (2-mercaptoethanol was added to reduced samples) and electrophoresed at 130 V, 20 mA for 1 h, 30 min. The gels were stained with 0.1% Naphthol Blue Black, 0.9 N acetic acid, and 20% ethanol for a minimum of 6 h and destained electrophoretically. The stained gels were subsequently scanned using a Zeneih soft laser microdensitometer (Biomed Instruments, Inc.). Fully reduced Reduction and oxidation of protamines in solution. protamine was prepared by dissolving the protein in 6 M guanidine hydrochloride (GuCl), 0.05 M Tris-HCl buffer, pH 8, 0.002 M ethylenediaminetetraacetate (EDTA) containing a 50-fold excess of dithiothreitol (DTT) and allowing the protein to reduce overnight (16 h minimum) at room temperature (23°C). The protein was then precipitated with trichloroacetic acid (added to a final concentration of 20%), washed with acidified acetone, and dried under nitrogen. Such samples had to be used immediately, since disulfide crosslinks begin forming at a slow rate as soon as the protein is dried and exposed to air. All experiments were performed with the proteins dissolved in 0.1 M Tris-HCl buffer, pH 8.0 at a protamine concentration of 1 mg/ml. Samples (l- to 2-ml aliquots) were incubated at 23°C in open 15-ml Corex tubes without agitation, unless otherwise noted. Disulfide crosslink formation was terminated (in every experiment except those containing sodium chloride, urea or guanidine hydrochloride) by adding 100% aqueous TCA to the sample to a final concentration of 20%, allowing the protein to precipitate on ice for 30 min, centrifuging, and washing the pellet three times with acidified acetone. Time intervals refer to the length of time between dissolving solid protamine in the appropriate buffer and the addition of TCA to precipitate it. The effects of sodium chloride, urea, and guanidine hydrochloride on the formation of intraprotsmine disulfides were determined by dissolving the reduced protamine in 0.1 M Tris-HCl, pH 8, containing the appropriate concentration of salt or denaturing agent and allowing the di-
3 Abbreviations used: TFA, trifluoroacetic drochloride; EDTA, ethylenediaminetetraacetate; TCA, trichloroacetic acid.
acid; GuCl, DTT,
guanidine hydithiothreitol;
DISULFIDES
IN
VITRO
385
sulfides to form for four hours. The pH was reduced to below pH 5 by adding an equal volume 0.1 N HCl, the samples were dialyzed against 10 mM HCl (twice), and the proteins were recovered by lyophilization or TCA precipitation. The catalytic effect of copper on protamine disulfide formation was determined by incubating reduced protamine in 0.1 M Tris-HCl, pH 8, containing 5 pM cuprous chloride (CuCl -2HaO) at 23°C. The protein samples were precipitated with TCA and analyzed. The effect of pH on disulfide formation was determined by dissolving freshly reduced, TCA precipitated bull protamine in 0.1 M Tris-HCl buffer titrated to the appropriate pH with either 5 N hydrochloric acid or sodium hydroxide. While Tris does not buffer well at extremes of pH, this buffer was used throughout the pH range to minimize potential anionic or cationic effects. The buffering capacity of Tris in this experiment proved to be sufficient to prevent a pH change greater than 0.2 pH units during the 2-h incubation period. After incubation at 23°C for 2 h in an open tube, the protamine was precipitated with TCA and the crosslinked forms analyzed by disc gel electrophoresis. Protamine (1 mg) Methyl&on and carboxymethyZ&ion of p&amine. was reduced with a 50-fold excess of DTT in 6 M GuCl, 0.1 M Tris-HCl, pH 8, and 2 mM EDTA for 1 h. The protein was methylated under nitrogen by adding a total of three 200-~1 aliquots of methyl iodide (57 rl/ml absolute ethanol) at 15-min intervals. The sample was subsequently dialyzed against 2 liters distilled water and lyophilized. Similar aliquots of protein were carboxymethylated after reduction with DTT by adding a twofold excess (over total sulfhydryl plus DTT) of sodium iodoacetate and reacting the samples for 2 h at 23°C in the dark under nitrogen. The protein was desalted and recovered by chromatography on a 1 X 30-cm Sepahdex G-25 column in 10 mM HCl and lyophilized. ProtHPLC separation of disulfide crosslinked forms of protamine. amine (3 mg) was dissolved in 5 ml 0.1 M Tris-HCl, pH 8, and allowed to fold at 23°C for 24 h. The protein was precipitated with TCA and redissolved in 0.1% TFA immediately prior to HPLC on a Nucleosil RP-Cl8 column (7.5 mm i.d. X 300 mm; Machery-Nagel, Duren, FRG). The different forms of folded protamine were separated using a linear gradient of acetonitrile. Buffer A contained 0.1% aqueous TFA. Buffer B contained 40% acetonitrile and 0.1% TFA. The gradient was run at a rate of 2 ml per minute, from 35 to 65% Buffer B, over a 40-min period. Protein elution was monitored at an absorbance of 220 nm using a Waters Model 450 detector (Waters Associates, So. San Francisco, CA). The collected fractions were divided into two equal aliquots and TCA precipitated. One sample was dissolved in electrophoresis buffer and the other was reduced with DTT as described above, TCA precipitated, and dissolved in the same buffer. Gel filtration chromatography of proposed dinners and monomers. Gel filtration chromatography of the proteins isolated in HPLC fractions 1 + 2 and 3 was performed on a 21.5 X 300-mm TSK G3000SWG column (Toyo-Soda Manufacturing Co., Japan) equilibrated with 0.1% TFA. The flow rate of the column was maintained at 2 ml/min and the eluting proteins were detected by measuring the absorbance at 214 nm. Seven other proteins and peptides were dissolved in 0.1% TFA and cochromatographed as molecular weight standards: calf thymus histone Hl, MW 22,130; lysozyme, MW 14,388 (Sigma Chemical Co., St. Louis, MO); salmine dimer produced by glutaraldehyde crosslinking (37), MW 8500; reduced bull protamine, MW 6627; salmon protamine, MW 4250 (Sigma Chemical Co., St. Louis, MO); and the synthetic peptides RsWGR,j (MW 2315) and (RF), (MW 1160). These last two peptides were synthesized by the Protein Structure Laboratory, University of California, Davis, California. DNA-protamine complenation. Calf thymus DNA (4 mg) was dissolved in 4 ml 0.1 M Tris-HCl, pH 8, overnight and sonicated to reduce its viscosity. Reduced bull protamine (4 mg dissolved in 10 ml 10 mM DTT, 0.1 M Tris-HCl, pH 8) was added and the sample allowed to sit for 30 min at 23’C. The precipitate was centrifuged for 10 min at 10,OOOg and washed in 0.1 M Tris-HCl, pH 8. After resuspending in 16 ml 0.1 M Tris-HCl, pH 8, the sample was divided into four equal aliquots and incubated at 23’C. The protamine was extracted from the DNA by adding
386
BALHORN,
CORZETT,
0.75 g solid GuCl, 2.81 g urea, and 1.1 g sodium chloride in 10.9 ml water. After sitting 30 min, 5 N HCl was added to a concentration of 0.5 N to precipitate the DNA. The sample was centrifuged at 4100g for 5 min and the supernatant containing protamine was dialyzed against 0.5 N HCl and 10 mM HCl, and then TCA precipitated. Isolation of folded protumine from bull sperm. Bull sperm were washed in 0.1 M Tris-HCl, pH 8, and centrifuged twice. The pellet was resuspended in 0.1 M Tris, pH 8, containing the appropriate amount of DTT to yield a DTT/protamine cysteine of 0.3 and incubated at 23’C for 1 h. After rinsing in pH 6 Tris-saline, the sperm were dissolved in GuCl and the protamine was extracted as described above. Special care was taken to make certain that the protein was always kept in solution at low pH to avoid spontaneous folding after isolation. These conditions had been checked previously using reduced protamine in solution; no folded forms were produced. RESULTS
Intraprotamine Disulfide Electrophoresis
Formation
as Visualized by Gel
Disc gel electrophoresis is used routinely to compare the apparent size and charge density of proteins. The acidurea system is well suited for separating basic proteins such as histones and protamines (35, 36). Fully reduced bull protamine migrates toward the cathode in this system as a single, well defined band (Fig. lA, bull protamine). In the presence of excess DTT or mercaptoethanol, the sulfhydryl groups remain reduced and the protamine molecules migrate (at pH 2.4) as if they were in an extended conformation. Within minutes after solubilization buffer at pH 8, of reduced protamine in 0.1 M Tris-HCl five new electrophoretic species are formed, each with an electrophoretic mobility greater than that of the reduced, extended protamine molecule (Fig. lA, folded forms). As they appear, the band corresponding to the extended, reduced protamine (Fig. lB, position marked with an arrow) disappears. With increasing time, the quantitative distribution of protein within each band shifts progressively toward increased mobility. Coincident with the appearance of these higher mobility bands, several bands of lower mobility also appear (Fig. 1B). These protein bands represent only 20-25% of the total protein, even after 8 days at pH 8, and their formation is protein concentration dependent. The higher the protein concentration in the sample, the greater the percentage of lower mobility species produced (data not shown).
HPLC
Separation
of New Electrophoretic
Species
Chromatography of folded bull protamine (incubated at 21°C for 24 h) on a Nucleosil RP-Cl8 column resulted in the fractionation of the mixture into two major peaks (Fig. 2). Electrophoresis of the lyophilized fractions revealed that the first peak contained the higher mobility (monomer) protein species while the second peak contained the lower mobility (dimer) species. Fraction 1, the ascending side of the first peak, was enriched in those species with smaller apparent molecular weights. Fraction 2, the peak and descending side of the same peak, was
AND
MAZRIMAS
a Unfolded
Bull Protamine Folded Forms
b Reduced
*iL
d
10 min
Ii9
24 hr
1
8 days
FIG. 1. Appearance of folded forms of bull protamine in solution. (A) Five folded forms of the protamine monomer are resolved upon electrophoresis. Electrophoresis was performed for 1.5 h at 130 V, 20 mA in lo-cm acid-urea disc gels (from left to right). (B) Fully reduced bull protamine was dissolved in 0.1 M Tris-HCl buffer, pH 8, and the appearance of folded forms monitored by electrophoresis (from right to left) in acid-urea gels (see Materials and Methods section for details). Microdensitometer scans of the gels are shown in each panel. The arrow marks the position of the extended (unfolded) protamine molecule.
enriched in the species with electrophoretic mobilities closer to that of the extended protamine. Since these enriched species are stable and do not redistribute to form the original pattern upon dissolution and electrophoresis, we have ruled out the possibility that the faster mobility species represent an equilibrium product of solvent-protein interactions. Following treatment with excess DTT at pH 8, the electrophoretic mobility of all three fractions returned to
FORMATION
OF
INTRAPROTAMINE
DISULFIDES
IN
VITRO
387
FIG. 2. HPLC separation of folded forms of bull protamine. The panel on the left shows the chromatogram obtained upon HPLC of incubated protamine (24 h) on a Nucleosil RP-Cl8 column as described in the Materials and Methods. The panels on the right, labeled fractions 1 through 3, show the electrophoretic patterns of the protein species present in each HPLC fraction. Total folded protamine is shown in the top right panel for comparison. The dashed scan shows the mobility of the proteins in each fraction after reduction with excess DTT at pH 8. In all three cases, this protein has an electrophoretic mobility identical to that of the fully extended protamine molecule.
that of the extended, fully reduced bull protamine molecule. In some experiments, such as the one shown in Fig. 2, the disulfides were incompletely reduced and a small fraction of the protein remained folded. Upon retreatment with DTT, the residual disulfides could be completely reduced and the bands eliminated. This demonstrates that the higher mobility speciesare intact protamine molecules that contain folded regions stabilized by intramolecular disulfides and not degradation products of protamine. The lower mobility speciesappear to represent dimers of these protamine monomers linked by intermolecular disulfides. Analysis of Disulfide-Crosslinked Forms by Gel Filtration Chromatography To confirm that the slower mobility proteins (isolated as fraction 3 by HPLC) and the faster mobility proteins (isolated as fraction 1 + 2) observed by electrophoresis are dimers and monomers of protamine, we have compared their rates of elution through a TSK G3000SWG gel filtration column with a series of similar proteins and peptides of known molecular weight. While this approach alone cannot be used to obtain a definitive molecular weight value for a protein, it can be used to obtain an estimated molecular weight and should be sufficiently
sensitive to discriminate between multimeric forms (dimers, trimers, etc.) of a protein. The standard proteins included calf thymus histone Hl, lysozyme, a salmon protamine dimer, salmon protamine, unfolded bull protamine, and two arginine-rich synthetic peptides RGWGR, and (RF),. The results of the chromatography, shown in Fig. 3, indicate that the proposed protamine dimer (HPLC fraction 3) elutes from the column as if it had a molecular weight of 13,539 daltons. This value is very close to that expected for a bull protamine dimer (13,254 daltons). The proteins isolated as HPLC fractions 1 and 2, on the other hand, elute from the column as a broad peak that overlaps the reduced bull protamine peak and extends to the salmon protamine peak. This result is consistent with the identification of these proteins as folded monomeric forms of bull protamine. Factors Affecting Interprotamine
Disulfide Formation
As one might expect, the formation of the intraprotamine disulfides is highly pH dependent (Fig. 4). The smaller, disulfide-crosslinked folded forms of protamine do not form in solution at or below pH 6. The small amount of folded protamine observed in the pH l-6 samples shown in Fig. 4 represents residual intraprotamine disulfides that
388
BALHORN,
CORZETT,
AND
MAZRIMAS
ions (this metal catalyzes the air oxidation of thiols to disulfides). Not only are the bulk of the monomeric species shifted toward the smallest forms by 4 hours, but nearly 40% of the molecules migrate as dimers or higher multimers. It is expected that the central, polyarginine-rich domain of bull protamine (residues 16-36) would not be as flexible as the ends of the molecule and this region of the protein should remain fully extended in solution to minimize interactions between the many adjacent, positively charged arginine residues. Under such conditions, only five of the possible intramolecular disulfide combinations (Fig. 7) would produce folded structures different enough in length to be discriminated using the gel system employed. Struc-
50
60
70
80
90
Elution Volume (ml) FIG. 3. Estimation of molecular weights for the disulfide crosslinked bull protamine molecules isolated in HPLC fraction 3 (protamine dimers) and fractions 1 t 2 (folded monomers) by gel filtration chromatography. Calf thymus histone Hl (l), lysozyme (2), salmine dimer (3), reduced bull protamine (4), salmon protamine (5), I&WGR6 (6), and (RF), (7). Chromatography was performed on a TSK G30OOSWG column in 0.1% TFA. Protein elution was monitored at 214 nm.
persisted in the original reduced sample. On occasion, residual disulfides remain after reduction or reform during storage and their complete conversion to free thiols requires repeated DTT treatment. As the pH is increased above pH 7, on the other hand, the formation of smaller crosslinked forms of protamine accelerates dramatically. This is consistent with the participation of disulfides in stabilizing the folded structures. Disulfide formation requires the ionization of the sulfhydryl group in cysteine and this is facilitated at alkaline pH. The participation of cysteine residues in stabilizing the formation of the smaller folded forms of bull protamine was confirmed by examining the effect of cysteine modification upon the appearance of the higher mobility forms of protamine (Fig. 5). Both carboxymethylation of protamine cysteines with sodium iodoacetate and methylation with methyl iodide completely eliminated the appearance of structures with a higher electrophoretic mobility. Only the extended protamine molecule could be detected following incubation of the modified protamines at 23’C and pH 8 for 24 h. The formation of the disulfide crosslinks that stabilize the folded forms of protamine in vitro appears to be catalyzed by air oxidation. These crosslinks occur readily in solution without the addition of enzymes or oxidizing agents. While folded forms appear rapidly in solution above pH 7, the rate of their formation is increased dramatically (Fig. 6) in the presence of cuprous
PH 8
-k-L PH 9
ILL pH 10 _iji
FIG. 4. Effect of pH on the appearance of folded protamine species. See Materials and Methods for details. The dashed line marks the electrophoretic mobility of the fully extended bull protamine molecule.
FORMATION
OF
INTRAPROTAMINE
Carbox y meth y latedk
_h,
of protamine cysteine residues on the FIG. 5. Effect of modification appearance of folded protamine species. Fully reduced protamine was modified with either sodium iodoacetate or methyl iodide and the modified proteins were incubated at pH 8 for 24 h in 0.1 M Tris-HCl buffer. Both carboxymethylation (top set of panels) and methylation (bottom set) prevented the appearance of folded forms.
tures that differ in the positioning of a disulfide between two adjacent cysteines (cys5,cys6 or cys38,cys39) would not exhibit sufficiently different electrophoretic mobilities to be resolved as discrete bands. Intramolecular disulfides readily form in protamine even after it has bound to DNA (Fig. 8). The electrophoretic mobilities of the monomers isolated from the complex are indistinguishable from those produced by free protamine. This observation supports the idea that the cysteine residues that participate in forming the disulfides must occur near the ends of the molecule (within the amino- and carboxy-terminal domains involving residues 1-15 and 37-50) and that only the central arginine-rich domain binds to DNA. This domain is believed to be the region of the molecule that binds to DNA (33) with the peptide backbone extended so each arginine residue can interact with a phosphate group in DNA. Intermolecular disulfide crosslinks also form within the complex; after 6 h, all of the bound protamine is crosslinked around DNA and cannot be dissociated by guanidine hydrochloride. Experiments conducted with boar and mouse protamine 1 demonstrate that these protamines also form intramolecular disulfides in solution (Fig. 9). As one might expect, however, the apparent size and number of electrophoretic species observed with mouse, bull and
DISULFIDES
IN
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389
boar protamine 1 differed for each type of protamine. This presumably correlates with the number and spatial distribution of cysteine residues within each protamine. Folded forms of salmine, on the other hand, are not observed. This was expected, since the protein lacks cysteine. Salmine contains predominantly arginine residues and should remain fully extended in solution. To assessthe effect of hydrophobic and electrostatic interactions on the formation of the intramolecular disulfides in bull protamine, the experiments were repeated in the presence of urea, guanidine hydrochloride, and sodium chloride (Fig. 10). Urea appeared to have little if any effect on the process at concentrations up to 8 M. While guanidine hydrochloride produced no effect up to a concentration of 4 M, some suppression of the formation of the smaller folded forms was detected at the highest concentration, 8 M. Sodium chloride could only be tested to a maximum concentration of 1 M. At 1 M salt, folding occurred unhindered (data not shown). Above this concentration, the bulk of the protamine aggregated and precipitated. Intramolecular Disulfides In Vivo In an effort to determine if protamines containing intramolecular disulfides could be isolated from bull sperm, saline washed sperm were treated with the lowest molar ratio of DTT to protamine cysteines (0.3) that wouldpermit the dissociation of protamine from DNA. The proteins were extracted with guanidine hydrochloride at pH 5, dialyzed and handled under acidic conditions to prevent
ymin !Jp& 1 FIG. 6. Effect of cuprous ions on disulfide formation and the appearance of folded protamine species in uitro. Reduced bull protamine was incubated in 0.1 M Tris-HCl buffer, pH 8, containing 5 pM cuprous chloride for the period of time shown in each panel. The dashed scan and lines mark the electrophoretic mobility of the fully extended (reduced) protamine molecule.
390
BALHORN,
CORZETT, Extended
Central
AND
MAZRIMAS
Domatn
COOH
COOH
COOH
COOH
COOH
COOH
FIG. ‘7. Schematic representation of possible folded bull protamine structures produced in vitro and stabilized by intramolecular disulfide crosslinks. Disulfide formation has been limited to contacts that are expected for the protein in solution under conditions in which the central polyarginine segment of the molecule (arg15-arg36) is forced into a reasonably rigid, extended conformation. Because the charge on the protein does not vary, the electrophoretic mobility of the various structures should be proportional to their Stokes radius.
spontaneous disulfide formation or interchange during the isolation steps. Subsequent electrophoresis of the isolated protein in acid-urea gels (Fig. 11) revealed that the bulk of the protein contains intramolecular and intermolecular disulfides (the electrophoretic pattern reverts to that characteristic of fully reduced bull protamine upon treatment with DTT) and that the various forms present appeared similar to those produced by prolonged folding of bull protamine in solution. Essentially all of the protein migrated with an electrophoretic mobility characteristic of folded monomers or dimers. Equal amounts of protein were extracted as monomer (48%) and dimer (49%). In a control experiment, completely reduced protamine, extracted and handled in an identical manner, remained unfolded (see Fig. 11, dashed line).
DISCUSSION
Mammalian protamines differ from the protamines of fish and marsupials in that they contain cysteine (lo20%). In mature bull sperm each cysteine residue participates in the formation of intra- and intermolecular disulfides that lock neighboring protamines together around the DNA helix (33, 34) and stabilize the final structure of the chromatin. Analyses of protamine disulfide formation in viva have indicated that approximately half of the disulfides formed in bull and mouse sperm represent intramolecular crosslinks that form in the testis and caput epididymis (22). Progressive reduction/modification experiments using bull sperm have shown that the bull protamine molecule is also folded in uiuo (34). Both ends of the molecule are folded in toward its center and locked
FORMATION
OF
INTRAPROTAMINE
1 hr
4 hr
0 hr
DISULFIDES
IN
VITRO
391
The formation of intraprotamine disulfide crosslinks occurs spontaneously in vitro and appears to be controlled only by kinetic factors that relate to the small size of the protein and the short distances that separate cysteine residues. Since the formation of these disulfides is not affected by urea, guanidine hydrochloride, or sodium chloride, neither intramolecular hydrophobic or electrostatic interactions appear to play a significant role in facilitating the process. Surprisingly, the formation of these disulfides and folded forms of protamine occurs unhindered even when the protein is bound to DNA. This would suggest that the DNA binding site on bull protamine must be limited to the highly charged, central polyarginine core of the molecule. If either the carboxyl- or amino-terminal regions of protamine bound to DNA, we would have de-
FIG. 8. Folding and formation of inter- and intramolecular disulfide crosslinks in bull protamine complexed with DNA. Fully reduced bull protamine was mixed with calf thymus DNA and allowed to fold and form disulfides as described under Materials and Methods. Samples were taken at 0, 1,4, and 6 h after washing and resuspending the insoluble DNA-protamine complex in Tris-saline at pH 8, the dissociable proteins were extracted, and protamine folding was assessed by electrophoresis in acid-urea gels. Microdensitometer scans of the stained gels are shown. The direction of electrophoresis is from the left to the right.
in place by a single disulfide bridge. The formation of these two intramolecular disulfides in vim begins in latestep spermatids shortly after the protamines are deposited onto DNA. The present studies demonstrate that similar disulfidestabilized folded protamines also form in vitro and suggest that the fundamental mechanisms responsible for the formation of these disulfides in vitro may be the same ones that operate in uiuo. Bull protamine is a small protein only 50 amino acids in length. At or near neutral pH and at low ionic strength (- 10e5h? in the gel system used here), 19 of the 22 amino acids in the central, arginine-rich domain of the molecule are positively charged and the electrostatic repulsion between adjacent arginine sidechains would be expected to restrict the flexibility of this region of the molecule. This is supported by infrared studies which show that similar sequences in the protamines of fish adopt an extended conformation in solution (38). While the length and extension of the polyarginine-like domain in bull protamine appears to prevent the formation of disulfide crosslinks between cysteine residues located in opposite ends of the molecule (only a subset of the possible disulfide crosslinked forms of protamine are found in solution), further studies are required before we can be certain that none of the cysteine residues in the central polyarginine-rich domain participate in forming disulfides.
Mow.
1
FIG. 9. Electrophoretic analysis of disulfide-stabilized folded forms produced by salmine, boar protamine, and mouse protamine 1. Each protein was reduced and allowed to fold under the same conditions described for bull protamine (see Materials and Methods). Microdensitometer scans are compared for the fully reduced proteins and proteins allowed to fold at pH 8 for 24 h.
392
BALHORN,
CORZETT,
tected a difference in the rate of intraprotamine disulfide formation or the types of folded forms produced. Protamine disulfide formation in vitro appears to be mediated by air oxidation. Trace metals, such as copper, catalyze the oxidation of sulfhydryls to disulfides (39-43) and accelerate the appearance of folded forms of protamine. Whether the formation of protamine disulfides in vivo is also mediated by air oxidation (or the lack of a reducing environment) remains unknown. Interprotamine disulfide crosslinks are also produced in vitro, just as they are in the epididymis. Protamine dimers are evident in oxidized samples of bull protamine in solution, while higher polymers (trimers, etc.) only appear in limited amounts, even after extensive oxidation. As observed in vivo, the protamine molecules in synthetic DNA-protamine complexes also eventually form interprotamine disulfide crosslinks that interlock the neighboring protamines and prevent their extraction from DNA. Very specific interprotamine disulfides are formed in vivo. While the disulfides formed in synthetic DNAprotamine complexes may be similar to those formed in vivo, the intermolecular disulfides formed between protamine molecules in solution probably represent all possible combinations. Clearly the inward folding of the amino- and carboxyterminal domains of bull protamine 1 and the formation of these intraprotamine disulfides in vitro do not represent “protein folding” as we normally think of the process that
AND
MAZRIMAS
DIMER
MONOMER
+
FIG. 11.
Microdensitometer scan of folded forms of hull protamine isolated from minimally reduced (DTT/protamine cys = 0.3) bull sperm (solid trace) and unfolded protamine treated under identical conditions (dashed trace). See Materials and Methods for experimental details.
has been described for ribonuclease (44, 45) and many other proteins (46-49). The five different folded forms of protamine produced in these experiments appear to represent all the possible combinations of intramolecular disulfides that can occur (with an extended central domain) and can be resolved by the electrophoretic technique. Only one of these folded structures can contain both the cys6cysl4 and cys39-cys47 intramolecular disulfides that are formed in bull sperm chromatin (34). While it is not known how protamine disulfide formation is regulated during the final stages of sperm chromatin maturation, both the results of these in vitro experiments and the observation that intra- and intermolecular disulfides are formed sequentially (22) suggest that some mechanism must exist for dictating which cysteine residues participate in the formation of disulfides at any particular time and for precluding those cysteines that form d&sulfides later. How this discrimination is accomplished is currently unknown. ACKNOWLEDGMENTS This work was performed under of Energy at Lawrence Livermore by Contract W-7405-ENG-48.
the auspices of the U.S. Department National Laboratory and supported
REFERENCES 8M
FIG. 10.
Effect on the formation The experimental The dashed profile fully reduced bull
8M
of urea and guanidine hydrochloride concentration of disulfide-stabilized folded forms of bull protamine. conditions are described under Materials and Methods. shows the electrophoretic pattern and mobility of protamine.
T., and Suzuki,
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1992
Formation of lntraprotamine
Disulfides in vitro’
Rod Balhorn,’ Michele Corzett, and J. A. Mazrimas Biomedical
Received
Division,
July
L-452, Lawrence
1, 1991, and in revised
form
Livermore
March
National
Laboratory,
Livermore,
California
94550
20, 1992
When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solublization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein’s cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the aminoand carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro. Q 1992 Academic PWS, 1110.
Protamines are small, highly charged proteins that package the DNA of most vertebrate sperm in a highly
condensed, genetically inactive state. While the elucidation of the primary sequences of several fish protamines (l-11) has revealed that these proteins are little more than polyarginine molecules interspersed with occasional uncharged or phosphorylatable amino acids, the protamines of mammals (11-21) are considerably more complex. Two very different protamines have been isolated from mammalian sperm. Protamine 1, the smaller of the two proteins, contains 50 amino acids and is nearly twice the size of many fish protamines. The second molecule, protamine 2, has been isolated from a variety of species (22) but only the proteins from mouse (23), human (11, 18, 24-26), stallion (27), and hamster (19, 28) sperm have been sequenced. These proteins are larger than protamine 1 and each has a composition and primary sequence that differs dramatically from the known sequences of protamine 1. Primary sequences have been determined for protamine 1 isolated from bull (12, 17), ram (14), boar (13), mouse (Xi), hamster (19), and human (16, 18, 26) sperm. Comparisons of these protein sequences have demonstrated that the majority of the amino acid sequence in each molecule is highly conserved. The amino-terminus of each protein begins with the same sequence, ala-arg-tyr-argcys-cys. The central half of protamine contains tandemly repeated polyarginine stretches and resembles the entire molecule of fish protamine, both in sequence and size. Unlike the protamines of fish, however, mammalian protamines contain cysteine residues that are intercrosslinked, locking adjacent protamines around the DNA molecule (29-33). These proteins also contain carboxyl- and aminoterminal peptide sequences that are relatively deficient in arginine and appear to interact with other protamines or sperm nuclear proteins.
1 This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any States Government or the University of California. The views and opininformation, apparatus, product, or process disclosed, or represents that ions of authors expressed herein do not necessarily state or reflect those its use would not infringe privately owned rights. Reference herein to of the United States Government or the University of California, and any specific commercial products, process, or service by trade name, shall not be used for advertising or product endorsement purposes. trademark, manufacturer, or otherwise, does not necessarily constitute ’ To whom correspondence should be addressed. or imply its endorsement, recommendation, or favoring by the United 384 All
Copyright 0 1992 rights of reproduction
0003-9861/92 $5.00 by Academic Press, Inc. in any form reserved.
FORMATION
OF
INTRAPROTAMINE
Studies in this laboratory have revealed that the aminoand carboxyl-terminal ends of each bull protamine bound to DNA in mature sperm must be folded in toward the center of the molecule and locked in place by a disulfide bond (34). During maturation of the sperm in the epididymis, over half of the cysteine residues in the molecule are crosslinked through intramolecular disulfide bridges (34). We describe studies with bull protamine which demonstrate that this protein rapidly forms disulfide-stabilized folded structures in solution and that these structures are similar to those isolated from synthetic DNAprotamine complexes and partially reduced bull sperm. MATERIALS
AND METHODS
Isokzttin and purification of bull protamine. Bull semen was purchased from the American Breeders Service, DeForest, Wisconsin. Boar semen was obtained from Swine Genetics International, Inc., Cambridge, Iowa. Bull, boar, and mouse sperm basic proteins were isolated from frozen semen or epididymal sperm (mouse) as previously described (35). Protamine was separated from the mixture and purified by HPLC on a Nucleosil RP-Cl8 column using the ion-pairing agent trifluoroacetic acid (TFA)3 and a linear acetonitrile gradient (17). The fractions containing bull protamine were collected and lyophilized. Gel electrophoresis. Reduced, modified, and folded protamine samples were analyzed by disc gel electrophoresis using lo-cm acid-urea gels (35,36). These 15% acrylamide gels were preparedwith 0.4% crosslmking and contained 2.5 M urea. Samples were dissolved in minimal volumes of 0.9 N acetic acid, 20% sucrose (2-mercaptoethanol was added to reduced samples) and electrophoresed at 130 V, 20 mA for 1 h, 30 min. The gels were stained with 0.1% Naphthol Blue Black, 0.9 N acetic acid, and 20% ethanol for a minimum of 6 h and destained electrophoretically. The stained gels were subsequently scanned using a Zeneih soft laser microdensitometer (Biomed Instruments, Inc.). Fully reduced Reduction and oxidation of protamines in solution. protamine was prepared by dissolving the protein in 6 M guanidine hydrochloride (GuCl), 0.05 M Tris-HCl buffer, pH 8, 0.002 M ethylenediaminetetraacetate (EDTA) containing a 50-fold excess of dithiothreitol (DTT) and allowing the protein to reduce overnight (16 h minimum) at room temperature (23°C). The protein was then precipitated with trichloroacetic acid (added to a final concentration of 20%), washed with acidified acetone, and dried under nitrogen. Such samples had to be used immediately, since disulfide crosslinks begin forming at a slow rate as soon as the protein is dried and exposed to air. All experiments were performed with the proteins dissolved in 0.1 M Tris-HCl buffer, pH 8.0 at a protamine concentration of 1 mg/ml. Samples (l- to 2-ml aliquots) were incubated at 23°C in open 15-ml Corex tubes without agitation, unless otherwise noted. Disulfide crosslink formation was terminated (in every experiment except those containing sodium chloride, urea or guanidine hydrochloride) by adding 100% aqueous TCA to the sample to a final concentration of 20%, allowing the protein to precipitate on ice for 30 min, centrifuging, and washing the pellet three times with acidified acetone. Time intervals refer to the length of time between dissolving solid protamine in the appropriate buffer and the addition of TCA to precipitate it. The effects of sodium chloride, urea, and guanidine hydrochloride on the formation of intraprotsmine disulfides were determined by dissolving the reduced protamine in 0.1 M Tris-HCl, pH 8, containing the appropriate concentration of salt or denaturing agent and allowing the di-
3 Abbreviations used: TFA, trifluoroacetic drochloride; EDTA, ethylenediaminetetraacetate; TCA, trichloroacetic acid.
acid; GuCl, DTT,
guanidine hydithiothreitol;
DISULFIDES
IN
VITRO
385
sulfides to form for four hours. The pH was reduced to below pH 5 by adding an equal volume 0.1 N HCl, the samples were dialyzed against 10 mM HCl (twice), and the proteins were recovered by lyophilization or TCA precipitation. The catalytic effect of copper on protamine disulfide formation was determined by incubating reduced protamine in 0.1 M Tris-HCl, pH 8, containing 5 pM cuprous chloride (CuCl -2HaO) at 23°C. The protein samples were precipitated with TCA and analyzed. The effect of pH on disulfide formation was determined by dissolving freshly reduced, TCA precipitated bull protamine in 0.1 M Tris-HCl buffer titrated to the appropriate pH with either 5 N hydrochloric acid or sodium hydroxide. While Tris does not buffer well at extremes of pH, this buffer was used throughout the pH range to minimize potential anionic or cationic effects. The buffering capacity of Tris in this experiment proved to be sufficient to prevent a pH change greater than 0.2 pH units during the 2-h incubation period. After incubation at 23°C for 2 h in an open tube, the protamine was precipitated with TCA and the crosslinked forms analyzed by disc gel electrophoresis. Protamine (1 mg) Methyl&on and carboxymethyZ&ion of p&amine. was reduced with a 50-fold excess of DTT in 6 M GuCl, 0.1 M Tris-HCl, pH 8, and 2 mM EDTA for 1 h. The protein was methylated under nitrogen by adding a total of three 200-~1 aliquots of methyl iodide (57 rl/ml absolute ethanol) at 15-min intervals. The sample was subsequently dialyzed against 2 liters distilled water and lyophilized. Similar aliquots of protein were carboxymethylated after reduction with DTT by adding a twofold excess (over total sulfhydryl plus DTT) of sodium iodoacetate and reacting the samples for 2 h at 23°C in the dark under nitrogen. The protein was desalted and recovered by chromatography on a 1 X 30-cm Sepahdex G-25 column in 10 mM HCl and lyophilized. ProtHPLC separation of disulfide crosslinked forms of protamine. amine (3 mg) was dissolved in 5 ml 0.1 M Tris-HCl, pH 8, and allowed to fold at 23°C for 24 h. The protein was precipitated with TCA and redissolved in 0.1% TFA immediately prior to HPLC on a Nucleosil RP-Cl8 column (7.5 mm i.d. X 300 mm; Machery-Nagel, Duren, FRG). The different forms of folded protamine were separated using a linear gradient of acetonitrile. Buffer A contained 0.1% aqueous TFA. Buffer B contained 40% acetonitrile and 0.1% TFA. The gradient was run at a rate of 2 ml per minute, from 35 to 65% Buffer B, over a 40-min period. Protein elution was monitored at an absorbance of 220 nm using a Waters Model 450 detector (Waters Associates, So. San Francisco, CA). The collected fractions were divided into two equal aliquots and TCA precipitated. One sample was dissolved in electrophoresis buffer and the other was reduced with DTT as described above, TCA precipitated, and dissolved in the same buffer. Gel filtration chromatography of proposed dinners and monomers. Gel filtration chromatography of the proteins isolated in HPLC fractions 1 + 2 and 3 was performed on a 21.5 X 300-mm TSK G3000SWG column (Toyo-Soda Manufacturing Co., Japan) equilibrated with 0.1% TFA. The flow rate of the column was maintained at 2 ml/min and the eluting proteins were detected by measuring the absorbance at 214 nm. Seven other proteins and peptides were dissolved in 0.1% TFA and cochromatographed as molecular weight standards: calf thymus histone Hl, MW 22,130; lysozyme, MW 14,388 (Sigma Chemical Co., St. Louis, MO); salmine dimer produced by glutaraldehyde crosslinking (37), MW 8500; reduced bull protamine, MW 6627; salmon protamine, MW 4250 (Sigma Chemical Co., St. Louis, MO); and the synthetic peptides RsWGR,j (MW 2315) and (RF), (MW 1160). These last two peptides were synthesized by the Protein Structure Laboratory, University of California, Davis, California. DNA-protamine complenation. Calf thymus DNA (4 mg) was dissolved in 4 ml 0.1 M Tris-HCl, pH 8, overnight and sonicated to reduce its viscosity. Reduced bull protamine (4 mg dissolved in 10 ml 10 mM DTT, 0.1 M Tris-HCl, pH 8) was added and the sample allowed to sit for 30 min at 23’C. The precipitate was centrifuged for 10 min at 10,OOOg and washed in 0.1 M Tris-HCl, pH 8. After resuspending in 16 ml 0.1 M Tris-HCl, pH 8, the sample was divided into four equal aliquots and incubated at 23’C. The protamine was extracted from the DNA by adding
386
BALHORN,
CORZETT,
0.75 g solid GuCl, 2.81 g urea, and 1.1 g sodium chloride in 10.9 ml water. After sitting 30 min, 5 N HCl was added to a concentration of 0.5 N to precipitate the DNA. The sample was centrifuged at 4100g for 5 min and the supernatant containing protamine was dialyzed against 0.5 N HCl and 10 mM HCl, and then TCA precipitated. Isolation of folded protumine from bull sperm. Bull sperm were washed in 0.1 M Tris-HCl, pH 8, and centrifuged twice. The pellet was resuspended in 0.1 M Tris, pH 8, containing the appropriate amount of DTT to yield a DTT/protamine cysteine of 0.3 and incubated at 23’C for 1 h. After rinsing in pH 6 Tris-saline, the sperm were dissolved in GuCl and the protamine was extracted as described above. Special care was taken to make certain that the protein was always kept in solution at low pH to avoid spontaneous folding after isolation. These conditions had been checked previously using reduced protamine in solution; no folded forms were produced. RESULTS
Intraprotamine Disulfide Electrophoresis
Formation
as Visualized by Gel
Disc gel electrophoresis is used routinely to compare the apparent size and charge density of proteins. The acidurea system is well suited for separating basic proteins such as histones and protamines (35, 36). Fully reduced bull protamine migrates toward the cathode in this system as a single, well defined band (Fig. lA, bull protamine). In the presence of excess DTT or mercaptoethanol, the sulfhydryl groups remain reduced and the protamine molecules migrate (at pH 2.4) as if they were in an extended conformation. Within minutes after solubilization buffer at pH 8, of reduced protamine in 0.1 M Tris-HCl five new electrophoretic species are formed, each with an electrophoretic mobility greater than that of the reduced, extended protamine molecule (Fig. lA, folded forms). As they appear, the band corresponding to the extended, reduced protamine (Fig. lB, position marked with an arrow) disappears. With increasing time, the quantitative distribution of protein within each band shifts progressively toward increased mobility. Coincident with the appearance of these higher mobility bands, several bands of lower mobility also appear (Fig. 1B). These protein bands represent only 20-25% of the total protein, even after 8 days at pH 8, and their formation is protein concentration dependent. The higher the protein concentration in the sample, the greater the percentage of lower mobility species produced (data not shown).
HPLC
Separation
of New Electrophoretic
Species
Chromatography of folded bull protamine (incubated at 21°C for 24 h) on a Nucleosil RP-Cl8 column resulted in the fractionation of the mixture into two major peaks (Fig. 2). Electrophoresis of the lyophilized fractions revealed that the first peak contained the higher mobility (monomer) protein species while the second peak contained the lower mobility (dimer) species. Fraction 1, the ascending side of the first peak, was enriched in those species with smaller apparent molecular weights. Fraction 2, the peak and descending side of the same peak, was
AND
MAZRIMAS
a Unfolded
Bull Protamine Folded Forms
b Reduced
*iL
d
10 min
Ii9
24 hr
1
8 days
FIG. 1. Appearance of folded forms of bull protamine in solution. (A) Five folded forms of the protamine monomer are resolved upon electrophoresis. Electrophoresis was performed for 1.5 h at 130 V, 20 mA in lo-cm acid-urea disc gels (from left to right). (B) Fully reduced bull protamine was dissolved in 0.1 M Tris-HCl buffer, pH 8, and the appearance of folded forms monitored by electrophoresis (from right to left) in acid-urea gels (see Materials and Methods section for details). Microdensitometer scans of the gels are shown in each panel. The arrow marks the position of the extended (unfolded) protamine molecule.
enriched in the species with electrophoretic mobilities closer to that of the extended protamine. Since these enriched species are stable and do not redistribute to form the original pattern upon dissolution and electrophoresis, we have ruled out the possibility that the faster mobility species represent an equilibrium product of solvent-protein interactions. Following treatment with excess DTT at pH 8, the electrophoretic mobility of all three fractions returned to
FORMATION
OF
INTRAPROTAMINE
DISULFIDES
IN
VITRO
387
FIG. 2. HPLC separation of folded forms of bull protamine. The panel on the left shows the chromatogram obtained upon HPLC of incubated protamine (24 h) on a Nucleosil RP-Cl8 column as described in the Materials and Methods. The panels on the right, labeled fractions 1 through 3, show the electrophoretic patterns of the protein species present in each HPLC fraction. Total folded protamine is shown in the top right panel for comparison. The dashed scan shows the mobility of the proteins in each fraction after reduction with excess DTT at pH 8. In all three cases, this protein has an electrophoretic mobility identical to that of the fully extended protamine molecule.
that of the extended, fully reduced bull protamine molecule. In some experiments, such as the one shown in Fig. 2, the disulfides were incompletely reduced and a small fraction of the protein remained folded. Upon retreatment with DTT, the residual disulfides could be completely reduced and the bands eliminated. This demonstrates that the higher mobility speciesare intact protamine molecules that contain folded regions stabilized by intramolecular disulfides and not degradation products of protamine. The lower mobility speciesappear to represent dimers of these protamine monomers linked by intermolecular disulfides. Analysis of Disulfide-Crosslinked Forms by Gel Filtration Chromatography To confirm that the slower mobility proteins (isolated as fraction 3 by HPLC) and the faster mobility proteins (isolated as fraction 1 + 2) observed by electrophoresis are dimers and monomers of protamine, we have compared their rates of elution through a TSK G3000SWG gel filtration column with a series of similar proteins and peptides of known molecular weight. While this approach alone cannot be used to obtain a definitive molecular weight value for a protein, it can be used to obtain an estimated molecular weight and should be sufficiently
sensitive to discriminate between multimeric forms (dimers, trimers, etc.) of a protein. The standard proteins included calf thymus histone Hl, lysozyme, a salmon protamine dimer, salmon protamine, unfolded bull protamine, and two arginine-rich synthetic peptides RGWGR, and (RF),. The results of the chromatography, shown in Fig. 3, indicate that the proposed protamine dimer (HPLC fraction 3) elutes from the column as if it had a molecular weight of 13,539 daltons. This value is very close to that expected for a bull protamine dimer (13,254 daltons). The proteins isolated as HPLC fractions 1 and 2, on the other hand, elute from the column as a broad peak that overlaps the reduced bull protamine peak and extends to the salmon protamine peak. This result is consistent with the identification of these proteins as folded monomeric forms of bull protamine. Factors Affecting Interprotamine
Disulfide Formation
As one might expect, the formation of the intraprotamine disulfides is highly pH dependent (Fig. 4). The smaller, disulfide-crosslinked folded forms of protamine do not form in solution at or below pH 6. The small amount of folded protamine observed in the pH l-6 samples shown in Fig. 4 represents residual intraprotamine disulfides that
388
BALHORN,
CORZETT,
AND
MAZRIMAS
ions (this metal catalyzes the air oxidation of thiols to disulfides). Not only are the bulk of the monomeric species shifted toward the smallest forms by 4 hours, but nearly 40% of the molecules migrate as dimers or higher multimers. It is expected that the central, polyarginine-rich domain of bull protamine (residues 16-36) would not be as flexible as the ends of the molecule and this region of the protein should remain fully extended in solution to minimize interactions between the many adjacent, positively charged arginine residues. Under such conditions, only five of the possible intramolecular disulfide combinations (Fig. 7) would produce folded structures different enough in length to be discriminated using the gel system employed. Struc-
50
60
70
80
90
Elution Volume (ml) FIG. 3. Estimation of molecular weights for the disulfide crosslinked bull protamine molecules isolated in HPLC fraction 3 (protamine dimers) and fractions 1 t 2 (folded monomers) by gel filtration chromatography. Calf thymus histone Hl (l), lysozyme (2), salmine dimer (3), reduced bull protamine (4), salmon protamine (5), I&WGR6 (6), and (RF), (7). Chromatography was performed on a TSK G30OOSWG column in 0.1% TFA. Protein elution was monitored at 214 nm.
persisted in the original reduced sample. On occasion, residual disulfides remain after reduction or reform during storage and their complete conversion to free thiols requires repeated DTT treatment. As the pH is increased above pH 7, on the other hand, the formation of smaller crosslinked forms of protamine accelerates dramatically. This is consistent with the participation of disulfides in stabilizing the folded structures. Disulfide formation requires the ionization of the sulfhydryl group in cysteine and this is facilitated at alkaline pH. The participation of cysteine residues in stabilizing the formation of the smaller folded forms of bull protamine was confirmed by examining the effect of cysteine modification upon the appearance of the higher mobility forms of protamine (Fig. 5). Both carboxymethylation of protamine cysteines with sodium iodoacetate and methylation with methyl iodide completely eliminated the appearance of structures with a higher electrophoretic mobility. Only the extended protamine molecule could be detected following incubation of the modified protamines at 23’C and pH 8 for 24 h. The formation of the disulfide crosslinks that stabilize the folded forms of protamine in vitro appears to be catalyzed by air oxidation. These crosslinks occur readily in solution without the addition of enzymes or oxidizing agents. While folded forms appear rapidly in solution above pH 7, the rate of their formation is increased dramatically (Fig. 6) in the presence of cuprous
PH 8
-k-L PH 9
ILL pH 10 _iji
FIG. 4. Effect of pH on the appearance of folded protamine species. See Materials and Methods for details. The dashed line marks the electrophoretic mobility of the fully extended bull protamine molecule.
FORMATION
OF
INTRAPROTAMINE
Carbox y meth y latedk
_h,
of protamine cysteine residues on the FIG. 5. Effect of modification appearance of folded protamine species. Fully reduced protamine was modified with either sodium iodoacetate or methyl iodide and the modified proteins were incubated at pH 8 for 24 h in 0.1 M Tris-HCl buffer. Both carboxymethylation (top set of panels) and methylation (bottom set) prevented the appearance of folded forms.
tures that differ in the positioning of a disulfide between two adjacent cysteines (cys5,cys6 or cys38,cys39) would not exhibit sufficiently different electrophoretic mobilities to be resolved as discrete bands. Intramolecular disulfides readily form in protamine even after it has bound to DNA (Fig. 8). The electrophoretic mobilities of the monomers isolated from the complex are indistinguishable from those produced by free protamine. This observation supports the idea that the cysteine residues that participate in forming the disulfides must occur near the ends of the molecule (within the amino- and carboxy-terminal domains involving residues 1-15 and 37-50) and that only the central arginine-rich domain binds to DNA. This domain is believed to be the region of the molecule that binds to DNA (33) with the peptide backbone extended so each arginine residue can interact with a phosphate group in DNA. Intermolecular disulfide crosslinks also form within the complex; after 6 h, all of the bound protamine is crosslinked around DNA and cannot be dissociated by guanidine hydrochloride. Experiments conducted with boar and mouse protamine 1 demonstrate that these protamines also form intramolecular disulfides in solution (Fig. 9). As one might expect, however, the apparent size and number of electrophoretic species observed with mouse, bull and
DISULFIDES
IN
VITRO
389
boar protamine 1 differed for each type of protamine. This presumably correlates with the number and spatial distribution of cysteine residues within each protamine. Folded forms of salmine, on the other hand, are not observed. This was expected, since the protein lacks cysteine. Salmine contains predominantly arginine residues and should remain fully extended in solution. To assessthe effect of hydrophobic and electrostatic interactions on the formation of the intramolecular disulfides in bull protamine, the experiments were repeated in the presence of urea, guanidine hydrochloride, and sodium chloride (Fig. 10). Urea appeared to have little if any effect on the process at concentrations up to 8 M. While guanidine hydrochloride produced no effect up to a concentration of 4 M, some suppression of the formation of the smaller folded forms was detected at the highest concentration, 8 M. Sodium chloride could only be tested to a maximum concentration of 1 M. At 1 M salt, folding occurred unhindered (data not shown). Above this concentration, the bulk of the protamine aggregated and precipitated. Intramolecular Disulfides In Vivo In an effort to determine if protamines containing intramolecular disulfides could be isolated from bull sperm, saline washed sperm were treated with the lowest molar ratio of DTT to protamine cysteines (0.3) that wouldpermit the dissociation of protamine from DNA. The proteins were extracted with guanidine hydrochloride at pH 5, dialyzed and handled under acidic conditions to prevent
ymin !Jp& 1 FIG. 6. Effect of cuprous ions on disulfide formation and the appearance of folded protamine species in uitro. Reduced bull protamine was incubated in 0.1 M Tris-HCl buffer, pH 8, containing 5 pM cuprous chloride for the period of time shown in each panel. The dashed scan and lines mark the electrophoretic mobility of the fully extended (reduced) protamine molecule.
390
BALHORN,
CORZETT, Extended
Central
AND
MAZRIMAS
Domatn
COOH
COOH
COOH
COOH
COOH
COOH
FIG. ‘7. Schematic representation of possible folded bull protamine structures produced in vitro and stabilized by intramolecular disulfide crosslinks. Disulfide formation has been limited to contacts that are expected for the protein in solution under conditions in which the central polyarginine segment of the molecule (arg15-arg36) is forced into a reasonably rigid, extended conformation. Because the charge on the protein does not vary, the electrophoretic mobility of the various structures should be proportional to their Stokes radius.
spontaneous disulfide formation or interchange during the isolation steps. Subsequent electrophoresis of the isolated protein in acid-urea gels (Fig. 11) revealed that the bulk of the protein contains intramolecular and intermolecular disulfides (the electrophoretic pattern reverts to that characteristic of fully reduced bull protamine upon treatment with DTT) and that the various forms present appeared similar to those produced by prolonged folding of bull protamine in solution. Essentially all of the protein migrated with an electrophoretic mobility characteristic of folded monomers or dimers. Equal amounts of protein were extracted as monomer (48%) and dimer (49%). In a control experiment, completely reduced protamine, extracted and handled in an identical manner, remained unfolded (see Fig. 11, dashed line).
DISCUSSION
Mammalian protamines differ from the protamines of fish and marsupials in that they contain cysteine (lo20%). In mature bull sperm each cysteine residue participates in the formation of intra- and intermolecular disulfides that lock neighboring protamines together around the DNA helix (33, 34) and stabilize the final structure of the chromatin. Analyses of protamine disulfide formation in viva have indicated that approximately half of the disulfides formed in bull and mouse sperm represent intramolecular crosslinks that form in the testis and caput epididymis (22). Progressive reduction/modification experiments using bull sperm have shown that the bull protamine molecule is also folded in uiuo (34). Both ends of the molecule are folded in toward its center and locked
FORMATION
OF
INTRAPROTAMINE
1 hr
4 hr
0 hr
DISULFIDES
IN
VITRO
391
The formation of intraprotamine disulfide crosslinks occurs spontaneously in vitro and appears to be controlled only by kinetic factors that relate to the small size of the protein and the short distances that separate cysteine residues. Since the formation of these disulfides is not affected by urea, guanidine hydrochloride, or sodium chloride, neither intramolecular hydrophobic or electrostatic interactions appear to play a significant role in facilitating the process. Surprisingly, the formation of these disulfides and folded forms of protamine occurs unhindered even when the protein is bound to DNA. This would suggest that the DNA binding site on bull protamine must be limited to the highly charged, central polyarginine core of the molecule. If either the carboxyl- or amino-terminal regions of protamine bound to DNA, we would have de-
FIG. 8. Folding and formation of inter- and intramolecular disulfide crosslinks in bull protamine complexed with DNA. Fully reduced bull protamine was mixed with calf thymus DNA and allowed to fold and form disulfides as described under Materials and Methods. Samples were taken at 0, 1,4, and 6 h after washing and resuspending the insoluble DNA-protamine complex in Tris-saline at pH 8, the dissociable proteins were extracted, and protamine folding was assessed by electrophoresis in acid-urea gels. Microdensitometer scans of the stained gels are shown. The direction of electrophoresis is from the left to the right.
in place by a single disulfide bridge. The formation of these two intramolecular disulfides in vim begins in latestep spermatids shortly after the protamines are deposited onto DNA. The present studies demonstrate that similar disulfidestabilized folded protamines also form in vitro and suggest that the fundamental mechanisms responsible for the formation of these disulfides in vitro may be the same ones that operate in uiuo. Bull protamine is a small protein only 50 amino acids in length. At or near neutral pH and at low ionic strength (- 10e5h? in the gel system used here), 19 of the 22 amino acids in the central, arginine-rich domain of the molecule are positively charged and the electrostatic repulsion between adjacent arginine sidechains would be expected to restrict the flexibility of this region of the molecule. This is supported by infrared studies which show that similar sequences in the protamines of fish adopt an extended conformation in solution (38). While the length and extension of the polyarginine-like domain in bull protamine appears to prevent the formation of disulfide crosslinks between cysteine residues located in opposite ends of the molecule (only a subset of the possible disulfide crosslinked forms of protamine are found in solution), further studies are required before we can be certain that none of the cysteine residues in the central polyarginine-rich domain participate in forming disulfides.
Mow.
1
FIG. 9. Electrophoretic analysis of disulfide-stabilized folded forms produced by salmine, boar protamine, and mouse protamine 1. Each protein was reduced and allowed to fold under the same conditions described for bull protamine (see Materials and Methods). Microdensitometer scans are compared for the fully reduced proteins and proteins allowed to fold at pH 8 for 24 h.
392
BALHORN,
CORZETT,
tected a difference in the rate of intraprotamine disulfide formation or the types of folded forms produced. Protamine disulfide formation in vitro appears to be mediated by air oxidation. Trace metals, such as copper, catalyze the oxidation of sulfhydryls to disulfides (39-43) and accelerate the appearance of folded forms of protamine. Whether the formation of protamine disulfides in vivo is also mediated by air oxidation (or the lack of a reducing environment) remains unknown. Interprotamine disulfide crosslinks are also produced in vitro, just as they are in the epididymis. Protamine dimers are evident in oxidized samples of bull protamine in solution, while higher polymers (trimers, etc.) only appear in limited amounts, even after extensive oxidation. As observed in vivo, the protamine molecules in synthetic DNA-protamine complexes also eventually form interprotamine disulfide crosslinks that interlock the neighboring protamines and prevent their extraction from DNA. Very specific interprotamine disulfides are formed in vivo. While the disulfides formed in synthetic DNAprotamine complexes may be similar to those formed in vivo, the intermolecular disulfides formed between protamine molecules in solution probably represent all possible combinations. Clearly the inward folding of the amino- and carboxyterminal domains of bull protamine 1 and the formation of these intraprotamine disulfides in vitro do not represent “protein folding” as we normally think of the process that
AND
MAZRIMAS
DIMER
MONOMER
+
FIG. 11.
Microdensitometer scan of folded forms of hull protamine isolated from minimally reduced (DTT/protamine cys = 0.3) bull sperm (solid trace) and unfolded protamine treated under identical conditions (dashed trace). See Materials and Methods for experimental details.
has been described for ribonuclease (44, 45) and many other proteins (46-49). The five different folded forms of protamine produced in these experiments appear to represent all the possible combinations of intramolecular disulfides that can occur (with an extended central domain) and can be resolved by the electrophoretic technique. Only one of these folded structures can contain both the cys6cysl4 and cys39-cys47 intramolecular disulfides that are formed in bull sperm chromatin (34). While it is not known how protamine disulfide formation is regulated during the final stages of sperm chromatin maturation, both the results of these in vitro experiments and the observation that intra- and intermolecular disulfides are formed sequentially (22) suggest that some mechanism must exist for dictating which cysteine residues participate in the formation of disulfides at any particular time and for precluding those cysteines that form d&sulfides later. How this discrimination is accomplished is currently unknown. ACKNOWLEDGMENTS This work was performed under of Energy at Lawrence Livermore by Contract W-7405-ENG-48.
the auspices of the U.S. Department National Laboratory and supported
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FIG. 10.
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