©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Diverse Effects of Mutation on the Activity of the Escherichia coli Export Chaperone SecB (*)

(Received for publication, March 24, 1995; and in revised form, June 27, 1995)

Harvey H. Kimsey (§) Mirabelle D. Dagarag Carol A. Kumamoto (¶)

From the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecBbulletpreMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecBbulletpreMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecBbulletpreprotein complexes at the membrane translocation site.


INTRODUCTION

The interaction of the export chaperone SecB with nascent or newly synthesized preproteins is an early event in Escherichia coli protein export(1) . SecB binding prevents the premature folding or aggregation of preproteins and maintains them in an export-competent conformation(2, 3, 4, 5) . SecB also greatly stimulates the rate of protein export, possibly through a ``targeting'' activity that involves conveying preprotein to the export apparatus in a specific conformation(6) . The best studied natural SecB ligand is maltose-binding protein (MBP), (^1)the periplasmic maltose receptor required by E. coli for maltose utilization(7) . SecB binds to nonoverlapping regions within a central portion of unfolded MBP(8) . Exported proteins that do not require the chaperone activity of SecB for export may interact with the GroEL-GroES or DnaK-DnaJ-GrpE heat shock chaperones prior to export(9, 10) .

There are differences between the SecBbulletpreprotein interaction and the interaction of the heat shock chaperones with their substrates. The heat shock chaperones have broad substrate-binding specificities, and they bind and hydrolyze ATP in order to release bound proteins(11) . In vitro, SecB binds with high affinity to a variety of peptides and unfolded proteins, many of which are not natural SecB substrates(12, 13) . SecB is not known to bind or hydrolyze ATP. Dissociation of SecBbullet preprotein complexes may involve preprotein binding to SecA, a peripheral membrane protein required for general protein export. SecA binds and hydrolyzes ATP (14) and, when membrane-bound, has a high affinity for SecBbulletproOmpA complexes formed in vitro(15) .

Mutational studies could be used to identify SecB residues that function in preprotein binding. Previously, chemically induced amino acid substitution mutations were shown to cluster in two regions of SecB(16, 17) . Mutations in region 1 were tightly clustered at positions Leu-75, Cys-76, and Glu-77 (Fig. 1). Mutations in the amino-terminal region 2 occurred at the acidic residues Asp-20 and Glu-24. Two region 1 mutants studied in detail, secBL75Q and secBE77K, are both strongly defective in promoting the rapid export of MBP from the cytoplasm. However, purified mutant SecB protein containing either alteration retains the ability to interact with unfolded MBP and prevent folding in vitro(16) . These two mutations cause SecB to exhibit apparently enhanced binding to unfolded MBP, suggesting that region 1 may play a role in preprotein binding. To better understand the roles of regions 1 and 2 in SecB function, a new set of mutations was generated in both regions. We describe here the effect these mutations exert on both the kinetics of MBP export and SecB:preMBP complex formation.


Figure 1: Single residue substitutions in SecB. The topsequence shows the wild-type SecB amino acid sequence. Columnsbelow represent single residue substitutions at each position. Mutations were generated using corresponding oligonucleotides containing low level random base substitutions (see ``Materials and Methods''). Mutants were divided into 3 classes using a colony color assay as a semi-quantitative measure of secB activity. Class I mutants were the least defective in secB activity, while Class III mutants were extremely defective in secB activity.




MATERIALS AND METHODS

Strains and Plasmids

The parent strain used in this work is the E. coli K-12 derivative MC4100 (F- DeltalacU169 araD139 rbsR rpsL thiA relA). CK1961 is MC4100 secB::Tn5 malT^czhe::Tn10 recA1. HK51 is MC4100 secB4 malT^cmalE18-1 srl::Tn10 recA1. HK57 is MC4100 secB::Tn5 malT^cmalE18-1 srl::Tn10 recA1. pHK202, a derivative of pBR322, lacks the tet gene and all sequences between the EcoRI site and the AvaI site and contains in addition an fd phage ori region for packaging single-stranded phage DNA. pHK205, the parent plasmid of the mutants described here, contains the 4.5-kilobase pair EcoRI fragment from pAK323(18) , carrying the normal secB gene, cloned into the unique EcoRI site of pHK202.

Site-directed Mutagenesis

The 34-mer oligonucleotide 5`-AAACCGCGTTCCTGTGTGAAGTTCAGCAGGGCGG corresponding to region 1, and the 40-mer 5`-TTTATACCAAGGATATCTCTTTCGAAGCGCCGAACGCGCC corresponding to region 2, were synthesized under conditions that should result in an average of 1.5 mutations/oligonucleotide molecule. This was accomplished by adding 5.88% (v/v) of an equimolar mixture of all four nucleotide precursors to each monomer phosphoramidite solution. Mixing proportions were determined from equations described by Ner and Smith(19) . Single-stranded templates of pHK205 were prepared in the dutungstrain CJ236 (20) using R408 helper phage. Phosphorylated mutagenic oligonucleotides were used to direct DNA synthesis as described previously(20) , except that T7 DNA polymerase holoenzyme was used in place of T4 DNA polymerase for the synthesis reaction(21) . Mutagenized plasmid pools prepared from these reactions contained greater than 1 times 10^4 members.

secB Mutant Screen

Mutagenized plasmid pools were introduced into HK51 by electroporation and plated on tetrazolium indicator agar (22) supplemented with 1.0% (w/v) maltose and ampicillin (100 µg/ml). The HK51 screening strain constitutively synthesizes preMBP(18-1), which contains an arginine substitution in the hydrophobic core of the signal sequence(23) , effectively blocking export of about 95% of total preMBP(24) . HK51 also contains a partially defective chromosomal secBL75Q gene, further impairing MBP export to the extent that the strain is unable to utilize maltose (Mal) and produces red colonies on maltose tetrazolium indicator agar. The secBL75Q mutation is efficiently complemented by a cloned wild-type secB gene resulting in partially restored MBP(18-1) export and Mal white colonies. Plasmid transformants carrying new secB mutations produced pink or red Mal colonies on indicator agar due to their failure to complement the chromosomal secBL75Q mutation. Transformants were screened directly for pink or red Mal phenotypes after growth at 30 °C. In these mutagenesis experiments, approximately 11-15% of total Amp^R transformants were Mal. Mutations were identified by DNA sequencing of the mutagenized region using Sequenase 2.0 (U. S. Biochemical Corp.) and procedures and reagents supplied with the enzyme. Prior to DNA sequencing of the region 2 mutants, extracts of candidate mutants were first examined for the presence of full-length SecB protein by Western blot analysis. To confirm that mutagenesis did not introduce other secB mutations, the entire secB sequence of the following 18 mutants was determined: F74I, F74Y, L75R, L75M, L75P, C76Y, E77V, E77A, V78F, Q79P, Q80R, D20H, D20A, D20Y, E24Q, E24D, E24A, E24V. In no case was a second mutation found.

Pulse-chase Analysis of MBP Export

secB mutant plasmids were introduced into CK1961 by electroporation. Cultures were grown in M63 media (22) supplemented with 0.2% (v/v) glycerol, 0.4% maltose, thiamine (0.5 µg/ml), MgSO(4) (10 mM), and carbenicillin (100 µg/ml). Pulse-chase experiments measuring preMBP export were carried out as described previously(5) . preMBP and MBP bands were quantitated using phosphorimaging methods, using software provided by the manufacturer (Molecular Dynamics). The fraction of total MBP in precursor form was calculated after adjusting for the fact that preMBP contains 9 methionines and mature MBP contains 6 methionines.

Preparation of Antisera

His-tagged SecB protein was purified from extracts of cells expressing a His-tagged secB derivative (^2)using procedures and plasmids provided by the manufacturer (Qiagen). Rabbits were immunized with His-tagged SecB protein as described previously(25) , except that Titermax(TM) was used as adjuvant. Anti-SecB antiserum was preadsorbed with an E. coli cell extract lacking SecB protein prior to use in immunoprecipitation experiments. Anti-MBP antiserum was prepared previously(25) .

Detection of SecBbulletpreMBP(18-1) Complexes

secB mutant plasmids were introduced into HK57 by electroporation. Cultures were grown at 37 °C in M63 minimal media as described above and labeled with 50 µCi of TranS-label/ml of culture for 5 min. 1-ml portions were transferred to chilled centrifuge tubes, and spheroplasts were prepared using egg white lysozyme (Sigma) as described previously(2) . Spheroplasts were collected by 30 s of centrifugation at maximum speed in an Eppendorf model 5415 microcentrifuge at 4 °C. The supernatant periplasmic fraction was discarded, and the spheroplasts were gently lysed in 0.5 ml of buffer I (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM Na(2)-EDTA, 2%(v/v) Triton X-100) by repeated pipetting. Protease inhibitor phenylmethylsulfonyl fluoride was immediately added to a final concentration of 0.1 mM. Extracts were centrifuged for 30-45 s at maximum speed in an Eppendorf model 5415 microcentrifuge at 4 °C, and 200-µl supernatant portions were removed and added to new tubes on ice containing 300 µl of buffer I and anti-SecB antisera or anti-MBP antisera. Immune complexes were formed for 1 h at 4 °C with constant mixing. To collect immune complexes, 50 µl of a slurry of protein A-Sepharose CL-4B beads (Pharmacia Biotech Inc.) (1:1 in buffer I) was added, and mixing was continued for 30 min. The beads were pelleted by centrifugation for 15-20 s at low speed in an Eppendorf model 5415 microcentrifuge at 4 °C and washed twice with 0.5 ml of cold high salt buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 1%Triton X-100) and once with 0.5 ml of 10 mM Tris-HCl, pH 8.0. Bound radiolabeled proteins were solubilized in Laemmli gel sample buffer. Electrophoresis on 12.5 or 15% SDS-polyacrylamide gels and fluorography with diphenyloxazole were carried out as described previously(5) . Gels were also analyzed using phosphorimaging detection plates. preMBP and SecB bands were quantitated using a Molecular Dynamics PhosphorImager. For the SecBbulletpreMBP stoichiometry measurements, radiolabeling was performed using TranS-label (ICN), which contains both [S]methionine and [S]cysteine. Calculations were carried out assuming uniform labeling of all cysteines and methionines. PhosphorImager data were adjusted for the fact that SecB contains 5 methionine and 4 cysteine residues and preMBP(18-1) contains 8 methionine residues.


RESULTS AND DISCUSSION

To further clarify the roles of region 1 and region 2, we generated a new collection of single-residue substitution mutants in both regions and characterized their effects on SecB function. To circumvent the limitations associated with chemical mutagenesis, mutations were targeted to region 1 and region 2 using synthetic oligonucleotides containing low level random base substitutions.

To identify secB mutants, we employed a red/white colony color assay that monitors the localization of MBP to the periplasm and thereby reflects cellular secB activity(26) . This assay relies on the expression of preMBP(18-1), a form of MBP containing a partially defective signal sequence. Using this assay, a mutant screen was set up to identify new secB mutants based on their failure to efficiently complement a chromosomal secBL75Q mutation. Employing this screen, 20 unique single residue substitutions were recovered in region 1 (Fig. 1). Small insertion or deletion mutations, as well as stop-codon mutations, were recovered at other positions within region 1, indicating that mutagenesis occurred throughout the targeted region. 10 unique substitutions were identified in region 2, all occurring at positions Asp-20, Ser-22, and Glu-24 (Fig. 1). A number of double, triple, and quadruple substitutions were also recovered in region 2. However, each of these contained at least one change at Asp-20, Ser-22, or Glu-24 (data not shown).

This collection of mutants was divided into three classes by estimating the levels of secB activity using the red/white colony color assay (Fig. 1). In these experiments, plasmids expressing mutant SecB supplied the only source of cellular SecB. Class I mutants retained substantial secB activity and included all of the substitutions at Phe-74, Cys-76, Val-78, plus E77D and S22A in region 2. Class II mutants showed a more severe secB deficiency and included most substitutions at Leu-75 and Glu-77 in region 1 and all of the substitutions at Asp-20 and Glu-24 in region 2. Three class III mutants, each the result of proline substitution, occurred in region 1. These mutants lacked secB activity completely because they failed to support growth of E. coli on rich media, a characteristic of E. coli strains devoid of SecB(27) . Western blot analysis of numerous region 1 and region 2 mutants revealed that only the proline substitutions in region 1 prevented high-level accumulation of SecB in growing cells (data not shown). Proline substitution in this region probably prevents normal folding, leading to rapid proteolytic degradation.

MBP Export Defects Associated with secB Mutants

The colony color assay results indicate that these mutants fail to efficiently export preMBP(18-1) to the periplasm. The export defect was also observed by examining the kinetics of normal MBP export in a subset of secB mutants. The extent and rate of MBP export was monitored indirectly by measuring the rate of preMBP signal peptide cleavage in pulse-chase experiments. As shown in Fig. 2A, substitutions in region 1 resulted in a wide range of kinetic defects in MBP export. The general pattern emerged that substitutions at Leu-75 and Glu-77 caused a severe slowing of the rate of MBP export, whereas changes at Phe-74, Cys-76, Val-78, and Gln-80 showed weak to moderate impairment of MBP export. Mutants F74Y and V78G, which emerged in our screen as phenotypically weak mutants, did not reliably show kinetic defects in MBP export (data not shown). In region 2, diverse substitutions at Asp-20 and Glu-24 all resulted in severe defects in MBP export, similar to that observed for substitutions at Leu-75 and Glu-77 (Fig. 2B). Overall, these results closely matched our mutant rankings obtained using the colony color assay.


Figure 2: Kinetics of maltose-binding protein export in secB mutant strains. secB mutant plasmids were introduced into strain CK1961 (malEsecB::Tn5 recA1). MBP export kinetics were determined by monitoring the rate of cleavage of the preMBP leader peptide. Cultures were pulse-labeled for 15 s with TranS-label as described. A pulse sample was taken, and the chase was begun by adding chloramphenicol (3.4 µg/ml) and unlabeled methionine (0.1 mg/ml) simultaneously. Chase samples were collected 30 and 60 s after the addition of chase. Samples were immunoprecipitated with anti-MBP antiserum and separated by SDS-polyacrylamide gel electrophoresis. Relative amounts of preMBP and mature MBP were determined using a Molecular Dynamics PhosphorImager; percent of total MBP in the precursor form is shown. Experiments were carried out in duplicate, and each bar represents the average of two measurements.



SecBbulletpreMBP Complex Formation

Since the defect in MBP export could result from the failure of SecB to associate with preMBP, region 1 and region 2 mutants were surveyed for their ability to form SecBbulletpreMBP complexes in living cells. SecBbulletpreMBP complexes were previously identified in cell extracts using immunoaffinity methods (25) . preMBP(18-1) was chosen as a substrate to examine complex formation because its defective leader peptide partially blocks preMBP export, leading to high intracellular preMBP levels that might favor complex formation. The altered leader peptide of preMBP(18-1) should not influence complex formation, since SecB binds to a central portion of unfolded MBP and binding is not affected by the presence of a signal peptide(8) .

PreMBP(18-1) was coimmunoprecipitated with SecB using anti-SecB antisera (Fig. 3A, lane2). Other SecB ligands, such as preLamB and proOmpA, were not detected because they contain functional signal sequences and are rapidly exported from the cytoplasm. PreMBP(18-1) did not fortuitously cross-react with SecB antibodies because it was not precipitated from extracts lacking SecB due to mutation (Fig. 3A, lane1). Co-precipitation of SecB and preMBP(18-1) was reduced if 5 µg of unlabeled purified competitor SecB was added to the extracts prior to the addition of antisera, indicating that SecB and preMBP(18-1) were present in these extracts as soluble complexes (Fig. 3B, lanes1 and 3). SecBbulletpreMBP(18-1) complexes were also detected when anti-MBP antisera was used in place of anti-SecB antisera (Fig. 3A, lane5).


Figure 3: Detection of SecB:preMBP(18-1) complexes in radiolabeled cell extracts. Extracts were prepared and incubated with anti-SecB antiserum or anti-MBP antiserum as described. Immune complexes were isolated and fractionated by SDS-polyacrylamide gel electrophoresis; fluorograms are shown. M, anti-MBP antiserum; B, anti-SecB antiserum. A, extracts were prepared from [S]methionine-labeled cells expressing SecB, preMBP(18-1), or both, as indicated by ± symbols. The relevant genotypes are as follows: lanes1 and 4, secB::Tn5 malE18-1; lanes2 and 5, secB::Tn5 malE18-1 pHK205(secB); lanes2 and 6, secB::Tn5 DeltamalB101 pHK205(secB). B, the secB::Tn5 malE18-1 pHK205(secB) strain was labeled with TranS-label. Extracts were prepared and immunoprecipitated as in panelA. For lane3, 5 µg of purified His-tagged SecB protein was added to the extract prior to incubation with anti-SecB antiserum. C, SecB:preMBP(18-1) complex formation in secB mutant strains. secB mutant plasmids were transformed into the secB::Tn5 malE18-1 strain HK57. The resulting strains were labeled with TranS-label. Extraction and immunoprecipitation were as in panelA.



The results of co-precipitation experiments with five region 1 mutants are shown in Fig. 3C. Complexes between SecBF74I and preMBP(18-1) were not detected using either anti-SecB or anti-MBP antisera (Fig. 3C, lanes1 and 2). Similarly, complexes involving SecBC76Y or SecBQ80R were observed at greatly reduced levels using either anti-SecB or anti-MBP antisera (Fig. 3C, lanes5 and 6 and lanes9 and 10). In contrast, complexes involving SecBL75R or SecBE77V were observed at levels comparable with that seen with normal SecB (Fig. 3C, lanes3 and 4 and lanes7 and 8).

While approximately 12-15% of the total precipitable preMBP(18-1) was observed in SecBbulletpreMBP complexes with normal SecB, most or all of the normal SecB exists in SecBbulletpreMBP(18-1) complexes (Fig. 3B, lanes1 and 2). Quantitation of these co-precipitation results yielded estimated SecBbulletpreMBP(18-1) stoichiometries of 4-5:1 for normal SecB, SecBL75R, and SecBE77V, indicating that SecBL75R and SecBE77V share the same binding stoichiometry as normal SecB. Since SecB is probably a tetramer(28) , the complexes we observe appear to be composed of one SecB tetramer and one preMBP(18-1) chain. Since each SecB monomer contains one peptide binding site(12) , SecBbulletpreMBP(18-1) complexes may consist of one SecB tetramer bound at multiple points along one preMBP chain.

Overall, region 1 mutants showed a distinct pattern of preMBP(18-1) binding defects (Fig. 4). Substitutions at alternating residues Phe-74, Cys-76, Val-78, and Gln-80 abolished or greatly reduced preMBP binding. In contrast, diverse substitutions at Leu-75 and Glu-77 all failed to disrupt preMBP complex formation. There are two exceptions to this pattern in region 1: SecBF74Y stably bound preMBP(18-1), whereas three Phe-74 substitution mutants involving the branched chain amino acids did not, indicating that an aromatic side chain at position 74 is essential for preMBP binding. Also, SecBV78G formed complexes with preMBP(18-1) but SecBV78F did not, indicating that only changes that introduce bulky side chains at Val-78 are sufficient to disrupt preMBP binding.


Figure 4: Summary of SecB:preMBP(18-1) co-precipitation results.



It is striking that mutations at Phe-74/Cys-76/Val-78/Gln-80 appear to strongly destabilize preMBP(18-1) binding but result in only mild defects in the rate of MBP export in living cells. During the course of our binding assay, preMBP(18-1) may continue to fold into a mature-like form that is not a substrate for SecB binding(13) . When a competing folding reaction removes preMBP substrate, binding defects due to lowered affinity could be greatly magnified, leading to an apparent all-or-none binding pattern. The high level synthesis of SecB, which occurs in these strains from multi-copy plasmids, may overcome or suppress export defects caused by a decreased affinity for preprotein.

These results suggest that the side chains at Phe-74/Cys-76/Val-78/Gln-80 are part of a hydrophobic preMBP binding pocket, or are required for the formation of such a binding pocket. The alternating pattern of binding-deficient mutations further suggests that region 1 is one strand of beta-sheet secondary structure. Region 1 could be one strand of a stable beta-sheet, analogous to the large beta-sheet that forms the floor of the peptide binding site in class I MHC protein, a protein that binds a wide variety of peptides(29) . This site may be identical to the hydrophobic site described by Randall as capable of binding the fluorescent compound 1-anilinonaphthalene-8-sulfonate(12) .

Intermixed with the binding site residues are Leu-75 and Glu-77. If region 1 is one strand of beta-sheet secondary structure, then the Leu-75 and Glu-77 side chains would project out onto the side opposite that displaying the Phe-74/Cys-76/Val-78/Gln-80 side chains. In such an arrangement, Leu-75 and Glu-77 are unlikely to participate in the same hydrophobic preprotein binding site. Consistent with this proposal, we found that diverse substitutions at both sites did not impair complex formation (Fig. 4). Nevertheless, changes at Leu-75 and Glu-77 resulted in more severe kinetic defects in MBP export than did changes at the binding site residues Phe-74/Cys-76/Val-78/Gln-80 (Fig. 2). Excluding the proline substitution at Leu-75, substitutions at Leu-75 and Glu-77 did not prevent high level accumulation of SecB in cells, indicating that changes at these positions do not grossly disturb SecB structure. Taken together, these observations indicate that Leu-75 and Glu-77 are essential for normal SecB function, but do not directly participate in preprotein binding.

Region 2 has several features in common with region 1. First, the alternating pattern of mutations suggests that region 2 also is composed of beta-sheet secondary structure. Second, changes at the acidic residues Asp-20 and Glu-24 did not disrupt preMBP(18-1) binding but resulted in slowed rates of MBP export similar to those associated with changes at Leu-75 and Glu-77 in region 1 (Fig. 2). Also, the two glutamyl residues Glu-24 and Glu-77 share the property that neither tolerates the conservative substitution by aspartic acid. An essential difference between the two regions is the apparent absence of binding site residues in region 2.

The overall similarities in mutant phenotypes caused by changes at Asp-20-Glu-24 and Leu-75-Glu-77 suggests that these residues function together in a SecB-mediated activity distinct from preprotein binding. Clues to the roles of Asp-20, Glu-24, Leu-75, and Glu-77 in SecB function come from previous biochemical studies of SecB protein altered at two of these positions. Purified SecB protein carrying the L75Q or E77K substitution retained the ability to interact with chemically unfolded MBP and prevent its rapid refolding(16) . In those studies, both mutant SecB proteins were more effective than normal SecB at blocking the refolding of MBP, indicating that certain alterations at Leu-75 and Glu-77 result in SecB with a higher affinity for unfolded MBP.

Residues Leu-75 and Glu-77, together with Asp-20 and Glu-24, may function to facilitate a conformational change leading to the opening of a closely associated hydrophobic preprotein binding site. Randall has reported evidence suggesting that positively charged regions of an unfolded preprotein are first bound at a site on SecB, and then a conformational change occurs and a second hydrophobic site on SecB becomes acccessible(12) . According to this two-step model of preprotein binding, Leu-75 and Glu-77 mutants could be locked in a normally short-lived high affinity conformation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM36415 (to C. A. K.) and was performed during the tenure of an American Heart Association Established Investigator Award (to C. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: 37 Manchester Rd., Newton, MA 02161.

To whom correspondence should be addressed: Dept. of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-0404; Fax: 617-636-0337.

(^1)
The abbreviations used are: MBP, maltose-binding protein; preMBP, precursor MBP.

(^2)
H. H. Kimsey and C. A. Kumamoto, unpublished results.


ACKNOWLEDGEMENTS

We thank Debabrata RayChaudhuri for good advice and warm collegiality. We also thank Pamela Gannon and Olivera Francetic for helpful discussions and members of the R. Isberg lab for technical advice.


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