the 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in...

The 19-residue pro-peptide of staphylococcal nucleasehas a profound secretion-enhancing ability inEscherichia coli

Dominic Suciu and Masayori Inouye *

Department of Biochemistry, Robert Wood JohnsonMedical School, Piscataway, New Jersey 08854, USA.

Summary

Staphylococcus aureus secretes two forms of extra-cellular nuclease, nuclease A and nuclease B. Nucle-ase A, consisting of 149 residues, is a proteolyticproduct of nuclease B, which is a processing inter-mediate that has a 19-residue N-terminal pro-peptidebetween the signal peptide and nuclease A. It hasbeen shown that nuclease A can be secreted byEscherichia coli by fusing it to the OmpA signal pep-tide. We now demonstrate that the addition of thepro-peptide between the OmpA signal peptide andnuclease A leads to a significantly enhanced secre-tion rate in E. coli . The processing and secretionrates of nuclease B at 37 8C were at least 10 timesfaster than those of nuclease A. Nuclease B wasalso secreted efficiently under conditions whichblocked the secretion of nuclease A, such as secAmutations and the addition of phenethyl alchohol orsodium azide. This enhancing effect of the pro-peptide was not as striking when it was attached tob-lactamase, indicating that the pro-peptide acts as aspecific secretion enhancer for nuclease A. Equili-brium circular dichroism on purified nuclease A andnuclease B indicated that the pro-peptide itself hadno significant destabilizing effect on the mature pro-tein. The existence of similar pro-peptides in Gram-positive bacterial secretory proteins indicates thatthey may also serve as secretion enhancers for indivi-dual proteins.

Introduction

A number of secretory proteins of Gram-positive bacteriahave been shown to be produced as pre-pro-proteins(Nagarajan, 1993). In addition to the signal peptide, thesesecretory precursors have N-terminal extensions,termed pro-peptides, between the signal peptide and the

mature protein, that play critical roles in the maturationof the protein (Nagarajan, 1993). Several roles havebeen envisioned for these pro-peptides: (i) in the case ofproteins such as subtilisin E of Bacillus subtilis (Ikemuraand Inouye, 1988) and a-lytic protease of Lysobacterenzymogenes (Silen and Agard, 1989), the pro-peptidefunctions as an intramolecular chaperone required forthe correct folding of the mature protein (for a review,see Shinde and Inouye, 1994); (ii) the pro-peptide of peni-cillinase of B. subtilis (Pollock, 1961; Lampen, 1967) hasbeen proposed to act as a temporary membrane anchorto the cell wall after signal-peptide cleavage; and (iii) it hasbeen proposed that some pro-peptides may enhancesecretion by acting either during translocation or duringinitial membrane insertion (Yamanaka et al., 1992;Wetmore et al., 1992).

Nuclease A is an exoenzyme of Staphylococcus aureuswhich is secreted into the culture medium. It is producedas a pre-pro-nuclease A which contains a 19-residuepro-peptide between the signal peptide and the matureregion (Shortle, 1983). The nascent secretory product ispro-nuclease A, which is designated nuclease B. Thepro-peptide of nuclease B is readily cleaved in vitro by aprotease from S. aureus strain V8 (Davis et al., 1977;1979). In vivo, nuclease B is processed to the mature Aform by the action of a phenylmethylsulphonyl fluoride(PMSF)-sensitive extracellular protease (Miller et al.,1987). In contrast to proteins such as the subtilisins, thepro-peptide of staphylococcal nuclease is not required forthe correct folding of the protein because, in its absence,enzymatically active nuclease A can be found in the peri-plasm and culture medium (Takahara et al., 1985; Milleret al., 1987; Liebl et al., 1992). Furthermore, it has beenshown that the kinetic parameters of refolding for nucleaseA and B are nearly identical (Davis et al., 1979).

Okabaiashi and Mizuno (1974) found that nuclease B isassociated with the cell wall of S. aureus, indicating thatthe pro-peptide may tether the protein to the cell wall.The proportion of A form to B form can be shifted bychanging the concentration of sodium chloride in the cul-ture medium; however, salt washes are ineffective inremoving cell-associated nuclease B (Liebl et al., 1992).Because of the unique features of the signal peptide ofstaphylococcal nuclease, namely the presence of twohighly hydrophobic stretches of approximately the same

Molecular Microbiology (1996) 21(1), 181–195

# 1996 Blackwell Science Ltd

Received 18 January, 1996; revised 8 April, 1996; accepted 10 April,1996. *For correspondence. Tel. (908) 235 4115; Fax (908) 235 4559.

g

length, separated by a hydrophilic region containing threebasic residues, it has been proposed that the pro-peptidemay modulate the function of this unusual signal peptide(Kovacevik et al., 1985).

In this study, we attempted to study the effect of the pro-peptide on the secretion of nuclease A by using an expres-sion system previously developed in this laboratory. In thissystem, nuclease A fused to the signal peptide of OmpA, amajor outer-membrane protein of Escherichia coli, can be

secreted by E. coli, generating fully active mature protein(Takahara et al., 1985). However, the secretion rate ofthis chimeric protein is not very efficient, and a largeamount of precursor pro-OmpA-nuclease A accumulatesin the cytoplasmic and membrane fractions of cells thatare overproducing this protein. In order to study the func-tion of the pro-peptide of staphylococcal nuclease, anexpression system producing a new chimeric protein,pro-OmpA-nuclease B, was constructed by inserting the

# 1996 Blackwell Science Ltd, Molecular Microbiology, 21, 181–195

182 D. Suciu and M. Inouye

nuclease B pro-peptide between the OmpA signal peptideand nuclease A. When this construct was expressed in E.coli, there was a dramatic increase in the secretion ofnuclease B. No accumulation of the precursor form, pro-OmpA-nuclease B, was observed, despite the fact thatthe protein is produced at approximately the same levelas pro-OmpA-nuclease A. Several methods for blockingsecretion, while blocking the secretion of pro-OmpA-nucle-ase A, had little effect on the secretion of pro-OmpA-nucle-ase B. Interestingly, the addition of the pro-peptide to pro-OmpA-b-lactamase did not show any enhancing effect onits secretion. From these results, we propose a possiblefunction of the pro-peptide as a specific enhancer of thesecretion of nuclease A.

Results

Expression of pro-OmpA nuclease A and B

The construction of pNuB, which contains the gene

expressing pro-OmpA-nuclease B, is shown in Fig. 1A.The expression and secretion into the periplasm of thepro-OmpA-nuclease hybrid protein have been shown tobe accompanied by the processing of the precursor pro-tein at the normal OmpA signal-peptide-cleavage site(Takahara et al., 1985). Pro-OmpA-nuclease A reachesa steady state of accumulation in the membrane fractionand in the inclusion bodies within the first 2 h of induction.Approximately 50% of the total production was in the formof the mature protein, almost 50% of which was found inthe culture medium (Fig. 2A). This distribution was main-tained until 9 h after induction (Fig. 2A, lanes 6 and 7).The slow processing of pro-OmpA-nuclease A into thesecreted mature protein appears to be very slow, resultingin the accumulation of a large amount of unprocessedprecursor.

The construction and sequence of pro-OmpA-nucleaseB is shown in Fig. 1A. When E. coli SB221 cells, harbour-ing pONF1, were induced with 2 mM IPTG to overexpress


Fig. 1. Construction of pNuB and pJGNB. Details of the constructions are given in the Experimental procedures.A. Construction of pNuB. pIN-III-OmpA3-#98 already contains six of the 19 amino acid residues from the pro-region of nuclease B. Theremaining 13 residues are supplied by Primer A. Primer B is the upstream primer. After PCR, a 160 bp fragment is created, containing theOmpA signal peptide fused to the remaining portion of the pro-peptide. This fragment is digested and ligated back into pIN-III-OmpA3-#98,generating pNuB.B. Construction of pJGNB. pJG105 is a pIN-III-OmpA3 derivative; therefore, the signal peptide and upstream region is the same as that ofpIN-III-OmpA3-#98 and pNuB. The same XbaI–BamHI OmpA signal peptide:nuclease B fragment from (A) is used to import the first 13residues from the pro-region. The remaining six residues are provided by a linker fragment. The actual ligation was performed as described inthe text, by first ligating in the nuclease B fragment.

Staphylococcal nuclease B contains a secretion-enhancing pro-peptide 183

pro-OmpA-nuclease A they showed a pronounced growthinhibition. However, cells harbouring pNuB and over-expressing pro-OmpA-nuclease B showed no such growthinhibition (data not shown). Under these conditions, onlya single band appeared at 1, 3, and 9 h after IPTGinduction (Fig. 2B), and this was also the case after 24 hof induction (data not shown). Interestingly, as can beseen from lane 5 and 7 in Fig. 2B, approx. 60% of totalproduct was secreted into the medium.

The fact that the induction of pro-OmpA-nuclease A istoxic to the cells whereas the production of pro-OmpA-nuclease B is not suggests that pro-OmpA-nuclease Bwas efficiently processed to form mature nuclease B, sothat toxic levels of pro-OmpA-nuclease B never accumu-lated in the cell. Therefore, the single band induced inFig. 2B is probably the processed mature nuclease B. Inorder to confirm this, we expressed pro-OmpA-nucleaseB in the E. coli strain IT41 which has temperature-sensitivesignal peptidase I activity (Inada et al., 1989). As can beseen in Fig. 3, in the strain IT41 the processing of pro-OmpA-nuclease B was significantly retarded at 428C,although not blocked completely. It is important to notethat no accumulation of pro-OmpA-nuclease B occurredwhen cells were grown beyond the chase time of 10 min.This result demonstrates that the single band seen inFig. 2B is indeed the processed mature nuclease B. It isinteresting to note that strain IT41 was unable to be trans-formed with pONF1. This is probably due to the fact thatleaky expression of pro-OmpA-nuclease A was enoughto accumulate a lethal amount of the precursor protein.The lower level of expression seen with strain IT41 at428C is due to the toxic effects of the inhibition of signalpeptidase I.

Figure 1A indicates that there is less production ofpro-OmpA-nuclease B than of pro-OmpA-nuclease A.This result is puzzling in light of the fact that both upstreamregions were found to have the same sequence. Oneconsideration is that nuclease B is completely secreted;therefore it is susceptible to degradation by extracellularproteases and normal protein degradation. In contrast,pro-OmpA-nuclease A accumulates inside the cell ininclusion bodies where, in an aggregated state, itis protected from digestion and degradation. Another


Fig. 2. Expression of Pro-OmpA-nuclease Aand Pro-OmpA-nuclease B in SB221. SB221cells harbouring pONF1 (A) and pNuB (B)were grown and processed as described inthe text. The supernatant (S) was precipitatedin 10% TCA, and processed as described inthe Experimental procedures. Samples weresubjected to SDS–PAGE, and the gel stainedwith Coomassie brilliant blue.

Fig. 3. Processing of pro-OmpA-nuclease B in strains IT42 andIT41. Cells of strains IT42 (lepA+) and IT41 (lepAts), a Spase Itemperature-sensitive mutant, were grown at 308C in M9 medium toa density of approx. 26108 cells ml71 (Klett 50). The cultures werethen transferred to 428C for 20 min. IPTG was then added to a finalconcentration of 2 mM. After a 20 min induction period, cells werelabelled with 10mCi ml71 of [35S]-methionine for 30 s. Cold methio-nine was then added and samples were withdrawn at the indicatedtime points. Each sample was precipitated with 10% TCA andimmune precipitation was performed using rabbit anti-nuclease.Gels were dried and exposed to Kodak XAR-5 film for 48 h. Aphotograph of the exposed film is shown. The bands correspondingto pro-OmpA-nuclease B (P), and nuclease B (NucB) are indicated.


possibility is that the pro-peptide may have some inhibitoryeffect during synthesis.

Effects of the pro-peptide on nuclease secretion

Results described above clearly indicate that pro-OmpA-nuclease B is more efficiently secreted than pro-OmpA-nuclease A. As can be seen in Fig. 4A, both the precursorform and the mature nuclease A appear at 378C even atthe pulse point (lane 1), while no precursor form is observedfor pro-OmpA-nuclease B. Instead, the B form appears initi-ally as a set of bands below that of the mature form, andthese bands are chased directly into the mature form ofthe protein. These bands are probably a result of translationpauses which allow nuclease-B secretion to occur co-trans-lationally. In contrast, pro-OmpA-nuclease A seems to be

processed both co-translationally (as indicated by theappearance of the mature band in the pulse lane) andpost-translationally as indicated by the gradual change inratio of upper (precursor) to lower (mature) bands over thetime-course of the experiment. With pro-OmpA-nucleaseB, when the temperature was lowered to 228C it becamepossible to detect a faint band appearing immediatelyabove that of the mature protein (Fig. 4B, lanes 7–10).Even this band is probably not the fully elongated precur-sor protein, as Fig. 3 indicated that the actual precursoris larger than this band. At 228C, the bands appearingbelow nuclease B are more intense and are still visibleeven at the 1 min point (lane 9); however, once again,most of this protein chases directly into the mature form.In contrast, pro-OmpA-nuclease A remains mostly in theprecursor even 40 min after synthesis, presumably in anaggregated form. These results indicate that whereas a


Fig. 4. In vivo processing of pro-OmpA-nuclease A and B, pro-OmpA-b-lactamase,and pro-OmpA-NucB-b-lactamase.In vivo processing of pro-OmpA-nuclease Aand B at 378C (A) and at 228C (B). Cellswere grown in M9 media to a density ofapprox. 26108 cells ml71 (Klett 50). IPTGwas then added to a final concentration of2 mM. After a 20 min induction period, cellswere labelled with 10mCi ml71 of [35S]-methio-nine for 30 s. Cold methionine was thenadded and samples were withdrawn at theindicated time points. Each sample was preci-pitated with 10% TCA and immune precipita-tion was performed using rabbit anti-nuclease(A and B) andanti-OmpA (A). Samples were processed andseparated by SDS–PAGE as described in thetext. Gels were dried and exposed to KodakXAR-5 film for 48 h. A photograph of theexposed film is shown. OmpA, a constitutivelyexpressed outer-membrane protein, was usedas an internal control for a comparison of theexpression of the two proteins. The bandscorresponding to OmpA (OmpA), nuclease A(NucA), pro-OmpA-nuclease A (P), andnuclease B (NucB) are indicated. In addition,cells harbouring pJG105 and pJGNB (C) werealso prepared at 378C as shown above. Thebands corresponding to b-lactamase (b-lac),and NucB-b-lactamase ([B]b-lac) areindicated. Precursor forms of both proteinsare the bands immediately above them.


majority of pro-OmpA-nuclease A was rendered transloca-tion incompetent by growth at 228C, pro-OmpA-nucleaseB became only partially post-translationally translocatedunder the same conditions.

As it appears that pro-OmpA-nuclease A is synthesizedin greater amounts than pro-OmpA-nuclease B, it maysaturate the secretion apparatus, leading to an accumula-tion of the A form. However, as can be seen in Fig. 4A,chromosomally expressed OmpA was not affected byexpression in cells overproducing either the A or B formof pro-OmpA-nuclease, indicating that the accumulationof pro-OmpA-nuclease A is not due to a general inhibitionof the secretion apparatus.

Next, we examined whether the 19-amino-acid-residuepro-peptide of nuclease B (pro-NucB) has a similar enhanc-ing effect on other secretory proteins. For this purpose,we constructed pJGNB, expressing pro-OmpA-pro-NucB-b-lactamase (see Fig. 1B), and compared its secretoryefficiency to that of pro-OmpA-b-lactamase, previouslyconstructed by Ghrayeb et al. (1984). Figure 4C showsa pulse-chase experiment using these two proteins. Thetwo hybrid proteins appear to be processed in the sameway as in both instances little accumulation of the precur-sor form is seen. The dramatic increase in processingrates seen with pro-OmpA-nuclease B is not reproducedwith pro-OmpA-(proB)-b-lactamase. This effect is probablydependent on the mature protein to which it is attached,and in this case seems to be specific to nuclease A. One

interesting observation is that the translation-pause band-ing patterns appear to be altered in the presence of thepro-peptide, suggesting that the pro-peptide may havesome effects on b-lactamase translation.

Cellular localization of the products

One concern with the pulse-chase data was that theycould have reflected merely an efficient processing rate,without a correspondingly efficient secretion rate. Inorder to determine whether the processed protein wasindeed secreted a cell fractionation was performed. Pro-OmpA-nuclease A accumulated in the cytoplasm whereasits processed form, nuclease A, appeared in the periplasmand in the culture medium (Fig. 5A). When cells wereconverted to sphaeroplasts and subjected to trypticdigestion, the mature translocated form was susceptibleto degradation, whereas the untranslocated precursorform was protected from tryptic digestion (Fig. 5C, lanes1–2). The processed form of pro-OmpA-nuclease B islocalized almost exclusively in the periplasm andmedium (Fig. 5B, lanes 2 and 5), indicating that nucleaseB was secreted across the inner membrane. This resultwas further confirmed by the results shown in lanes 3and 4 (Fig. 5C) in which sphaeroplasts were treatedwith trypsin; nuclease B was completely sensitive totrypsin.


Fig. 5. Cell fractionation and trypsin susceptibility of pro-OmpA-nuclease A and B. Cells harbouring pONF1 (A) and pNuB (B) were labelledwith [35S]-methionine for 30 s, followed by a 10 min chase as described in the Experimental procedures. The cells were then converted tosphaeroplasts by osmotic shock and divided into two aliquots. The first, the control (WCF), was simply incubated on ice. The second wasseparated into periplasmic (Peri), cytoplasmic (Cyt), and membrane (Mem) fractions. The culture medium (Sup) was treated with TCA. Atrypsin protection assay is shown in (C). From the samples described above, the third aliquot (Tryp) was treated with trypsin for 30 min at aconcentration of 0.5 mg ml71. Trypsin-treated samples were loaded in parallel to untreated control samples (WCF). All samples weresubjected to SDS–PAGE. Gels were dried and exposed to Kodak XAR-5 film for 48 h. Photographs of the exposed film are shown. The bandscorresponding to nuclease B (NucB), pro-OmpA-nuclease A (P), and nuclease A (NucA) are indicated.


Characterization of the purified forms of nuclease Aand B

Both nuclease A and B were purified to homogeneity.Activity assays on the two proteins were performed bythe method of Cuatrecassas et al. (1967). The specificactivities of nuclease A and nuclease B were 1130 and960 U mg71 respectively (standard deviation from themean was approx. 10% for both proteins). As has beenshown previously (Davis et al., 1977; Okabaiashi andMizuno, 1974), the extra 19-residue pro-peptide had littleeffect on the activity of nuclease B.

It has been shown previously (Cover et al., 1987; Liu etal., 1988; Teschke et al., 1991; Randall and Hardy, 1986)that there exists a positive correlation between compe-tence for secretion and a lack of tertiary structure. Thedestabilization of the mature protein was thought toincrease the time in which the polypeptide is competentfor export, thus making it a better candidate for efficientsecretion. Therefore, it is important to examine whetherthe pro-peptide of nuclease B would have an analogouseffect by destabilizing nuclease A. In order to answerthis question, equilibrium circular dichroism (CD) mea-surements of both the nuclease A and nuclease B formswere performed. Figure 6 shows the near- and far-UVspectra for nuclease A and nuclease B. The difference inthe far-UV spectra can be attributed to the extinction coef-ficient which takes into account the pro-peptide. An analy-sis of the far-UV spectra by the method of Sreerama andWoody (1993) indicates that there was no appreciablecontribution of the pro-peptide to the secondary structureof nuclease B. The near-UV spectra of the two proteinsare almost identical. This indicates that the pro-peptidehas no appreciable effect on the tertiary structure of nucle-ase A. The slightly lower minima at 278 nm and the peak

shift at 262 nm may be a contribution from the tyrosine inthe pro-peptide.

In order to study the relative stability of these two pro-teins, they were subjected to thermal denaturation. Theloss of secondary structure was monitored by measuringellipticity at 222 nm. The two melting transitions appear


Fig. 6. Far-UV (200–250 nm) and near-UV(250–320 nm) CD spectra of nuclease A andB. CD spectra for nuclease A (circles), andnuclease B (crosses) are shown. Proteinswere in 10 mM sodium phosphate, pH 7.0.Spectra were taken at 208C. Mean residue(200–250 nm) ellipticity and molar (250–320 nm) ellipticity are given in degreescm72 dmol71. Although plotted on the sameaxis, the values refer to the ellipticity frombackbone (far-UV) and aromatic (near-UV)contributions; therefore, the two spectra arenot to be considered continuous.

Fig. 7. Temperature dependence of the ellipticity of nuclease Aand B, as measured at 222 nm. Protein solutions were in 10 mMTris-HCl, pH 7.0, in a 1 mm rectangular cell. The temperatureincrease was either 1 or 28C per data point. Conversion of rawellipticity data to part unfolded values is performed by scaling theresults from 0 to 1. The y-axis refers to the position of eachmeasurement relative the native (0) and unfolded (1.0) states foreach transition. Empty symbols were used for nuclease A, filledsymbols were used for nuclease B. Each type of symbol representsa different trial for a given protein. At least two different trials wereplotted for each protein. The line that is plotted through the pointsrepresents the averaged results of the curve fitting for each dataset. It is the ideal curve for the entire data set, and it is from thesecurves that the values for standard free energy (DG) and meltingtemperature are derived.


to be superimposable (Fig. 7). The melting temperaturesfor nuclease A and B were 323.6 ±2.3 K (50.68C) and323.9 ±2.2 K (50.98C), respectively. A determination ofthe standard free energy of unfolding from these meltingcurves indicates that the difference between both thefree energies of unfolding and the melting temperature ofnuclease B and nuclease A is not statistically significant.The standard free energies of unfolding (DG) for nucleaseA and B were 5.0 ±1.6 kcal/mol and 5.7 ±0.3 kcal/mol,respectively. The standard free energy of unfolding deter-mined by Shortle and Meeker (1989) was 75.6 kcal/mol.It is therefore unlikely that the pro-peptide destabilizes thesecondary structure of nuclease A. An investigation wasalso performed in order to determine the effects of thepro-peptide on tertiary structure. Comparison of near-UVspectra of the two proteins taken at a series of tempera-tures indicated that there was no significant difference inbehaviour between the A and B form of nuclease (datanot shown).

Effect of phenethyl alcohol on the secretion andprocessing of pro-OmpA-nuclease A and B

Phenethyl alcohol (PEA) has been found to be destructiveto the permeability of the cell membrane. Cells treatedwith 0.3% PEA become permeable to nucleoside tri-phosphates, and protein synthesis in these cells relieson external addition of ATP and MgCl2 (Halegoua andInouye, 1979; Halegoua et al., 1976). Protein exportis an energy-driven process which requires both ATPhydrolysis and an electrochemical gradient across theinner membrane (Pugsley, 1993). Therefore, it is interest-ing to examine whether the pro-peptide was able tocontinue to have its enhancing activity in a cell in whichcellular permeability has disrupted the proton-motiveforce. In Fig. 8, A and B, one can see the effect of

PEA on cells expressing pro-OmpA-nuclease A and B. InFig. 8A, a mutant, pro-OmpA-nuclease A (F15W-W140F),was used which is secreted more efficiently than pro-OmpA-nuclease A because of the W140F mutation. Thisincrease in secretion efficiency of the mutant is a resultof a destabilization of the native structure of nuclease A(S. Chatterjee, D. Suciu, M. Inouye, in preparation). Thisefficiently secreted protein was used so that a moredramatic difference could be seen between the processingof the A and B forms. Lanes 1–4 of Fig. 8, A and B showthe protein expression of untreated cells. Note that pro-OmpA-nuclease (F15W-W140F) was completely pro-cessed to nuclease A because of the mutation. In lanes5–6, the protein expression of PEA-treated cells isshown. During the 3 h incubation with PEA, there was nocell growth as measured by the optical density of the cul-ture (data not shown). In lanes 7–9, protein expressionof PEA-treated cells grown in the presence of ATP andMgCl2 is shown. In the presence of ATP and MgCl2,there was a noticeable increase in the optical density.The ability of externally added ATP and MgCl2 to affectcell growth indicates that the permeability of the mem-brane was indeed achieved by PEA treatment. InFig. 8A one can see that PEA treatment completelyblocked the processing of pro-OmpA-nuclease A (F15W-W140F). However, as shown in Fig. 8B, no effect on pro-cessing can be seen on pro-OmpA-nuclease B, indicatingthat the pro-peptide is able to very effectively complementthe defect of protein secretion caused by PEA.

The role of SecA in the secretion and processing ofpro-OmpA-nuclease B

In addition to interacting with the membrane-bound pro-teins of the translocation apparatus, SecA has recentlybeen shown to act as an ATP-dependent protein pump


Fig. 8. Expression of pro-OmpA-nuclease A(F15W-W140F) and pro-OmpA-nuclease B inPEA-treated cells. pONF1- (A) and pNuB- (B)transformed E. coli cells were grown at 378Cin M9 media to a density of approx. 26108

cells ml71 (Klett 50), at which time 2 mMIPTG was added and the cultures were splitinto three fractions. The first was the controlfraction (lanes 1–3). The second fraction,labelled IP, was treated with 0.3% PEA (lanes5 and 6). The third fraction, labelled IPAM,was treated with 0.3% PEA, 25 mM ATP, and20 mM MgCl2. At each time point, cells werepelleted by centrifugation, resuspended insodium phosphate buffer, and sonicated.Samples were then subjected to SDS–PAGE.Sample volumes for each lane were inverselyproportional to the absorbance at 600 nm ofthe culture at that time point. The figure is aphotograph of a gel stained with Coomassiebrilliant blue.


during secretion (Economou and Wickner, 1994; Kim etal., 1994). In order to determine the role of SecA in thesecretion of pro-OmpA-nuclease B, two different methodsfor disrupting its activity have been used. First, sodiumazide, a potent and specific inhibitor of SecA in vivowhich specifically blocks the ATPase activity of SecA(Rusch et al., 1994), was added to cells expressingpro-OmpA-nuclease A and B. Figure 9A shows theresults of a pulse-chase experiment of cells treated withsodium azide. As can be seen in lanes 1–6, processing

of pro-OmpA-nuclease A was completely inhibited bysodium azide treatment, whereas the processing of pro-OmpA-nuclease B was hardly affected. It is interesting tonote that treatment with sodium azide leads to the appear-ance of translation-pause bands below pro-OmpA-nuclease A, and to a persistence of these bands for pro-OmpA-nuclease B.

The secretion of endogenously expressed OmpA pro-tein was monitored as an internal control. By comparingthe normal processing of pro-OmpA in Fig. 2A and theprocessing in the presence of sodium azide (Fig. 9A),one can clearly see the appearance of a precursor bandfor the OmpA protein in the first two lanes of the sodiumazide pulse-chase experiment, indicating that OmpAprocessing was inhibited. Figure 9B shows a cell fraction-ation performed on cell cultures at the end of the pulse-chase experiment. In lane 2, one can see that nucleaseA hardly appeared in the periplasm and that the over-whelming majority of nuclease A was in the form of pre-cursor, which was protected by the inner membranefrom trypsin treatment (lanes 4–5). In the case of pro-OmpA-nuclease B, processing had taken place and theprotein appeared in the periplasm (lane 7), where it wassensitive to trypsin treatment (lane 9). These resultsclearly indicate that sodium azide had little effect on thesecretion and processing of pro-OmpA-nuclease B.

Next, in order to determine whether pro-OmpA-nucle-ase B is still secreted when the levels of SecA proteinare decreased, a genetic approach was undertaken todisrupt SecA synthesis. Pro-OmpA-nuclease A and Bwere expressed in two strains bearing temperature-sensi-tive secA mutants. MM52 is temperature sensitive for theformation of active SecA, while MM66 is a temperature-sensitive secA amber mutant (Oliver and Beckwith, 1981;1982). At 428C, when compared to MC4100, approx.90% of pro-OmpA-nuclease A processing was blocked inboth of these mutant strains (Fig. 10A). In contrast, theprocessing of pro-OmpA-nuclease B was once again unaf-fected by the secA mutations. As it is known that in bothsecA mutant strains SecA assembled in the membranebefore the temperature shift and therefore did not loseits function even at the higher temperature, a residualSecA activity present under the current conditionsmay account for the complete processing of pro-OmpA-nuclease B.

Discussion

We have demonstrated that the 19-residue pro-peptideof staphylococcal nuclease B functions as a secretionenhancer of nuclease A when these proteins are secretedin E. coli using the OmpA signal peptide. Furthermore, incontrast to the secretion of pro-OmpA-nuclease A, thesecretion of pro-OmpA-nuclease B does not seem to be


Fig. 9. Effect of sodium azide on the processing and secretion ofpro-OmpA-nuclease A and B.A. In vivo processing of pro-OmpA-nuclease A and B in thepresence of 2 mM sodium azide. Pulse-chase experiments wereperformed at 378C. Cells were grown and induced as indicated inthe text. Each sample was precipitated with 10% TCA andimmunoprecipitated using rabbit anti-nuclease and anti-OmpAserum. Gels were dried and exposed to Kodak XAR-5 film for 48 h.A photograph of the exposed film is shown. OmpA, a constitutivelyexpressed outer membrane protein, was used as an internal controlfor a comparison of the expression of the two proteins. The bandscorresponding to OmpA (OmpA), nuclease A (NucA), pro-OmpA-nuclease A (P), and nuclease B (NucB) are indicated.B. Cell fractionation of sodium azide-treated SB221 cells expressingpro-OmpA-nuclease A and pro-OmpA-nuclease B was performedon the cultures in (A) at the 10 min time point, as described in theExperimental procedures. WCF refers to the whole cell fraction.Peri refers to the periplasmic fraction. M/C is the combinedmembrane/cytoplasmic fraction. T is the trypsin-treatedsphaeroplast fraction. TT is the trypsin- and Triton-X-treatedsphaeroplast fraction.


affected by SecA activity or by the presence of an electro-chemical potential across the inner membrane. In addition,a thermodynamic and structural characterization of puri-fied nuclease A and B indicated that there was no appre-ciable effect of the pro-peptide on the native structureof nuclease A, indicating that the pro-peptide was notincreasing secretion efficiency by slowing the folding rateof the secretory precursor. As the pro-peptide wasunable to enhance the secretion of b-lactamase when itwas part of the chimeric protein pro-OmpA-NucB-b-lacta-mase, these results indicate that the pro-peptide is prob-ably a specific enhancer for nuclease A, and that thisspecific secretion-enhancing effect of the pro-peptide isnot generated simply from the combination of the OmpAsignal peptide and the pro-peptide.

There are several possible explanations for this behav-iour: as was indicated in the Introduction, Gram-positivebacteria synthesize secretory precursors with pro-pep-tides which, among other things, have been found toenhance the secretion rate. The exact mechanism bywhich this occurs is still unknown. A similar pro-sequenceis found on the small ribonuclease barnase from Bacillusamyloliquefaciens. In a study by Gray et al. (1993) it wasfound that although this sequence has no effect on the invitro refolding of the protein, it mediates the associationof the the mature protein with GroEL. A similar role canbe envisioned for the pro-peptide of staphylococcal nucle-ase B, because it is conceivable that a preferrential

interaction with a chaperone could increase the secretionefficiency. The appearance of the translation-paused bandsmay be associated with the translocation efficiency. A par-tially synthesized protein may be more readily recognizedby a chaperone molecule such as SecB (Randall, 1992)and would be more easily routed into the translocationapparatus.

Another possiblity is suggested by an analysis of theN-terminal region of nuclease A. The loop model, originallyproposed by Inouye and Halegoua (1980), postulates thatthe signal peptide forms a loop with the mature protein as itinserts into the cellular membrane. This model requirescertain structural and charge distributions in the signalpeptide and in the mature region immediately adjacent toit. The N-terminal ends of signal peptides in E. coli containbasic amino acid residues which are thought to be local-ized on the cytoplasmic side of the membrane duringtranslocation (Gennity et al., 1990). On the other hand, astatistical analysis of 15 secretory proteins of E. coli indi-cates that the total charge of the first 10 residues of theN-terminus of the mature domain is 70.03, i.e. almostneutral (Yamane and Mizushima, 1988). From a statisticalstudy on a wide variety of bacteria, von Heijne (1986)pointed out that this domain is rather acidic. In fact, thepresence of positive charges in this region can stronglyinhibit secretion (Nilsson and von Heijne, 1990; Geller etal., 1993; Yamane and Mizushima, 1988). The first 10amino acid residues of nuclease A have a net charge of+2, having three lysines and one aspartic acid in thisregion. The first lysine appears at position +5 and +6from the cleavage site. Geller et al. (1993) found that inthe case of pro-OmpA, positively charged residues haveto be within two residues from the cleavage site in orderto inhibit secretion. However, one cannot discount the pos-sibility that the unusually high overall charge at the N-term-inal region of nuclease A may inhibit membrane insertionand secretion efficiency, either by stably interacting withthe negatively charged polar head groups of the mem-brane phospholipids or by interfering with insertion of themature region into the hydrophobic centre of the cyto-plasmic membrane. Indeed, the data presented here indi-cate that nuclease A is a rather poor secretory proteinwhen it is directly connected to the OmpA signal peptide.

The N-terminal sequence of the pro-peptide is ratherunique, having 13 hydrophilic residues and only 3 hydro-phobic residues (see Fig. 1). It is acidic, having 3 acidicand 1 basic residue. It is also interesting to note thatthere are two glycine residues at the 6th and 11th positionsof the pro-peptide. We propose that the addition of the pro-peptide in front of nuclease A offsets the inhibitory effect ofthe positively charged N-terminal region of nuclease A.However, the pro-peptide seems to have not only thesimple role of offsetting the inhibitory N-terminal region,but also a significant enhancing effect on nuclease A


Fig. 10. Processing of pro-OmpA-nuclease A and B in bacterialstrains MC4100, MM52, and MM62. Cells expressing pro-OmpA-nuclease A (A) and pro-OmpA-nuclease B (B) were grown at 308Cin M9 medium to early log phase, at which time cultures weretransferred to a 428C water bath for 2 h. Cells were then inducedwith 2 mM IPTG for 20 min, and a pulse-chase experiment wasperformed. Samples were withdrawn at the indicated timepoints. Each sample was precipitated with 10% TCA andimmunoprecipitated using rabbit anti-nuclease serum. Gels weredried and exposed to Kodak XAR-5 film for 48 h. A photograph ofthe exposed film is shown. The bands corresponding to nuclease A(NucA), pro-OmpA-nuclease A (P), and nuclease B (NucB) areindicated.


secretion, as processing of pro-OmpA-nuclease B wasseen both in the absence of SecA activity and in theabsence of an electrochemical gradient across the innermembrane. These facts may indicate that the initial mem-brane insertion may be independent of both SecA and ofthe presence of the electrochemical gradient. It appearsthat the initial insertion and processing of the signal pep-tide of the OmpA itself can take place in the absence ofa proton-motive force (Tani et al., 1989). The fact thatthe N-terminal regions of nuclease B and of the OmpA pro-tein are both neutral indicates that the pro-peptide func-tions by keeping the positively charged N-terminal end ofnuclease A away from the putative loop formed betweenthe signal peptide and the mature protein during mem-brane insertion (Halegoua and Inouye, 1979). In thisway, pro-OmpA-nuclease B would not be faced with thethermodynamic cost of inserting positively chargedamino acid residues into the hydrophobic centre of thecytoplasmic membrane, and membrane insertion cantake place, even in vivo, in the absence of SecA activityand of the electrochemical gradient. In contrast, in orderfor pro-OmpA-nuclease A to be inserted into the cytoplas-mic membrane, both these elements represent an abso-lute requirement.

Only a few proteins are known that do not require a func-tional sec pathway for translocation. Interestingly, theseproteins all have several membrane-spanning hydro-phobic regions (Gallusser and Kuhn, 1990; Rohrer andKuhn, 1990; von Heijne, 1989; Nilsson et al., 1993) withtranslocated segements that are less than 50 residues(Andersson and von Heijne, 1993). In a study by Anders-son and von Heijne (1993) it was proposed that proteinsthat are sec-independent may enter the sec pathway viaan initial non-ATP-dependent interaction with SecA, whereSecA would play a role solely in initial targetting. The find-ing that pro-OmpA-nuclease B is secreted under condi-tions in which SecA activity is inhibited may indicate thateither the initial targetting’ or the effective insertion ofthe signal peptide into the inner membrane can have afar greater influence on subsequent secretion steps thanpreviously anticipated.

Experimental procedures

Materials

The Sculptor mutagenesis kit was purchased from Amer-sham. All restriction endonucleases were from New EnglandBiolabs. Sodium azide was purchased from Sigma.

Bacterial strains, plasmids, and media

E. coli strain SB221 (lpp hsdR DtrpE5 leuB6 lacY recA1/F 'lacI q lac+ pro+) (Takahara et al., 1985) was used as hostcells for pro-OmpA nuclease expression, purification, and

pulse-chase experiments. E. coli strains MC4100 (F7(lac)U169 araD136 relA rpsL thi ), MM52 (MC4100, ts51)(Oliver and Beckwith, 1981), and MM66 (MC4100, secAam

Tn10 su 3ts trpam) (Oliver and Beckwith, 1982) were kindlyprovided by Donald Oliver of Wesleyan University. E. colistrains IT41(W3110 lep-9 TetR) and IT42(W3110 lep+

TetR) (Inada et al., 1989) were kindly provided by YoshikazuNakamura of the University of Tokyo. Plasmid pONF1 con-tains the nuclease A gene of S. aureus fused with theOmpA signal peptide (Takahara et al., 1985). pONF1(F15W-W140F) is a double mutant of pONF1 bearing aphenylalanine-to-tryptophan mutation at position 15 of theOmpA signal peptide and a tryptophan-to-phenylalaninemutation at position 140 of nuclease A (S. Chatterjee, D.Suciu, M. Inouye, unpublished). Plasmid pJG105 containsthe b-lactamase gene fused to the OmpA signal peptide(Ghrayeb et al., 1984). Cells were grown in M9 medium asdescribed previously (Inouye et al., 1976). For the pulse-chase experiments, casamino acids were replaced with 19amino acids (20mg ml71) (all except for methionine). For theexperiments with pONF1 and pNuB, the M9 medium contained50mg ml71 ampicillin. For the experiments with pJG105 andpJGNB, M9 medium contained 10mg ml71 of chloramphenicolin addition to ampicillin.

Construction of pNuB

Figure 1A depicts the method that was used to insert the 19-residue pro-peptide between the signal peptide and nucleaseA. pIN-III-OmpA3-#98, which contains the OmpA signal pep-tide fused to nuclease A and a portion of the pro region(Takahara et al., 1985; Ghrayeb et al., 1984), was used asa template for the polymerase chain reaction (PCR) in orderto generate a new XbaI–BamHI fragment. Oligonucleotideswere synthesized as described previously (Ghrayeb et al.,1984). PCR was performed using the following two primers:primer A, 5 '-GCTACCGTAGCGCAGGCCTCACAAACAGA-TAACGGCGTAAATAGAAGTGGTTCTGAGGATCCAAGT-3';and primer B, 5'-ATGACCATGATTAC-3'. The fragment gene-rated by PCR contained the OmpA signal peptide fused to themissing residues of the pro-peptide. After digestion with XbaIand BamHI, this region was ligated back into pIN-III-OmpA3-#98 creating pNuB which contained the OmpA signal peptidefused to the 19-residue pro-peptide and nuclease A.

Construction of pJGNB

Figure 1B describes the strategy used in inserting the19-residue pro-peptide of staphylococcal nuclease betweenthe OmpA signal peptide and mature b-lactamase. PlasmidJG105 (Ghrayeb et al., 1984) is actually a version of pIN-III-ompA3-#98, which contains the b-lactamase gene insertedin-frame at the EcoRI site, and a gene for chloramphenicolresistance inserted into the Pst I site of the ampicillin-resis-tance gene of this plasmid. JG105 was digested with XbaIand BamHI. Next, the 123 bp XbaI–BamHI fragment frompNuB was subcloned into pJG105, generating p19NB,which was chloramphenicol resistant. Next, a Bst EII–BamHI digestion product of JG105, containing the b-lacta-mase gene, was ligated into the BamHI site of p19NB using



a BamHI–Bst EII linker containing the missing residues of thepro-peptide. The linker was made using the following two pri-mers: primer C, 5 '-GTGACTATATACTGTTG-3 '; and primerD, 5 '-GATCCAACAGTATATA-3'. Annealing was performedas described previously (Ghrayeb et al., 1984). Transformedcells could be selected for the proper insertion of both DNAfragments by resistance to ampicillin. Figure 1B is a simplifieddescription of this procedure. This strategy was pursued inorder to have a better selection mechanism for the three-piece ligation step. All constructs were confirmed by sequenc-ing using the dideoxy chain-termination method of Sanger etal. (1977).

Expression of pro-OmpA nuclease

Cultures of cells harbouring pONF1 or pNuB were grown at378C to a density of approximately 26108 cells ml71 (Klett50) at which time IPTG was added to a final concentrationof 2 mM. At various induction times (t = 1, 3, and 9 h), Klettreadings were taken and a 1.5 ml sample was withdrawnfrom the culture. This sample was centrifuged for 5 min at90006g on a table-top centrifuge. Next, the upper 0.9 mlwas removed to another tube containing 100ml of 100% (w/v) trichloroacetic acid (TCA) and this supernatant fractionwas left on ice overnight. The TCA pellet was washed with0.5 ml acetone. After centrifugation and removal of acetone,the pellet was rewashed with 0.5 ml of ether. The washedpellet was dried and then resuspended by a brief sonicationin 30ml of 10 mM sodium phosphate buffer, pH 7.0. Next,7.5ml of 56SDS loading buffer (56SDS: 5% SDS, 25% gly-cerol, 200 mM b-mercaptoethanol, and 200 mM Tris-HCl,pH 6.8) was added to each sample.

The cell pellet was resuspended in 50ml of 10 mM sodiumphosphate buffer (pH 7.0). The samples were then sonicatedfor 60 s in a Heat Systems bath sonicator. A volume of 12.5mlof 56SDS loading buffer was then added to each sample. Allsamples were incubated for 5 min in a boiling-water bathbefore SDS–polyacrylamide gel electrophoresis (SDS–PAGE, 17.5% acrylamide concentration) was performed.Equal volumes of culture supernatant and whole cell fractionswere loaded for each time point. Sample loading volumeswere inversely proportional to the Klett reading for any giventime point.

Pulse-chase experiments

For pulse-chase labelling experiments, cultures were grownand induced with IPTG as indicated above. Next, each culturewas labelled with 10mCi ml71 of [35S]-methionine (AmershamCorp.; >1000 Ci mmol71). After 30 s of pulse labelling, non-radioactive methionine was added to a final concentration of40mg ml71 and a 0.9 ml pulse-sample (P) was removed andadded to a tube containing 100ml of 100% (w/v) TCA. Afterthis, chase samples were removed at the indicated times(see figure legends). They were processed in the samemanner as culture-supernatant samples above. Immune pre-cipitation was carried out as previously described using rabbitanti-nuclease antiserum (Inouye et al., 1976).

Cell fractionation

Cells were prepared as for the pulse-chase experiment

above; however, at the 10 min time point, cells were collectedby centrifugation (5 min at 50006g). Cells were first con-verted to sphaeroplasts by the method of Koshland andBotstein (1980) and Heppel (1967). After microscopic exam-ination to confirm that sphaeroplasts had indeed formed, eachsample was split into three fractions. The first was the controlfraction. To the second and third, trypsin was added to aconcentration of 0.5 mg ml71. To the third, Triton-X100 wasalso added to a concentration of 0.02%. The three sampleswere incubated on ice for 30 min, after which time a trypsininhibitor was added to a concentration of 0.5 mgml71. Duringthe digestion, a portion of the control fraction was removed.The sample was spun at 50006g for 5 min at 48C. The super-natant obtained was the source of the periplasmic fraction.The pellet was resuspended in an original volume of 10 mMsodium phosphate, pH 7.0, and sonicated as before for 30 s.In some experiments (Fig. 9B), the procedure was stoppedat this point and the sample was labelled ‘M/C’. The sampleswere then centrifuged for 30 min at 100 0006g in a BeckmanTLA 100.3 rotor in a Beckman TL100 tabletop ultra-centri-fuge. The resulting supernatant was the cytoplasmic fraction.The pellet, which was resuspended in an original volume ofphosphate buffer and sonicated, was the membrane fraction.Every sample was maintained in the original volume that wastaken from the control fraction at the beginning of the experi-ment. SDS–PAGE (17.5% acrylamide concentration) wasperformed on all samples. The gels were dried and exposedto Kodak XAR-5 film for 3 days.

Phenethyl alcohol and sodium azide treatment ofcells expressing pro-OmpA-nuclease A and B

E. coli SB221 cells transformed with pONF1(F15W-W140F)and pNuB were grown as described above. When the cellshad grown to a density of approx. 26108 cells ml71 (Klett50), they were induced with 2 mM of IPTG for 20 min. The cul-tures were then separated into three 10 ml fractions; the firstwas the control. To the second and third fraction, phenethylalcohol (Sigma) was added to a final concentration of 0.3%.To the third fraction, MgCl2 was added to a concentration of10 mM, and ATP was added to a concentration of 25 mM.Samples were taken at 0, 1, 2, and 3 h after induction and sub-jected to SDS–PAGE as described above.

Cells transformed with pONF1 and pNuB were grown andinduced as described above. After 15 min of induction theywere treated for 30 min with 1.0 mM sodium azide as pre-viously described (Rusch et al., 1994). Pulse-chase experi-ments and membrane fractionation were performed asdescribed above.

Effects of temperature-sensitive secA and lepA onnuclease secretion

pONF1 and pNuB were transformed in MC4100 (the controlstrain), MM52 (a temperature-sensitive secA) and MM66 (anamber mutant of secA) (Oliver and Beckwith, 1981; 1982).Cells were grown at 308C to a density of approx. 26108 cells ml71 (Klett 50), and cultures were then shifted to428C and grown for 2 h. Cultures were then inducedfor 20 min with 2 mM IPTG. At this point a pulse-chase



experiment was performed as described above. Sampleswere taken at 2 and 10 min.

For the temperature-sensitive signal peptidase I (SPase I)strain IT41, only pNuB was used as no colonies of pONF1-transformed cells could be isolated. IT41 and IT42 weretransformed with pNuB. When cultures had reached a densityof approx. 26108 cells ml71 (Klett 50), the cultures were placedin a 428C bath for 20 min followed by a 20 min induction with2 mM IPTG. Samples were collected at 0, 1, 5, and 10 min.

Purification of nuclease A and nuclease B

Purification of both nuclease A and B was performed on cellpellets from a 10 l culture induced with 2 mM IPTG for 10 h.The purification method was performed according to Shortleand Meeker (1989). An additional step had to be added at theend of the procedure in order to fully purify these proteins(Chatterjee et al., 1995). Protein was solubilized in 7.2 Mguanidine-HCl containing 3 mM b-mercaptoethanol and50 mM sodium citrate, pH 3.0. The samples were then dia-lysed against 15% glycerol containing 1 mM ethyleneglycol-bis-(b-aminoethyl ether) N,N,N ',N '-tetraacetic acid (EGTA)(Sigma), and 25 mM Tris-HCl, pH 8.0, followed by dialysisagainst 2 l of 25 mM HEPES, pH 7.5, with two changes. Therefolded protein was loaded on an S-Sepharose column(5361 cm) equilibrated with 25 mM HEPES, pH 7.5. Chroma-tography was performed on a Pharmacia fast-protein liquidchromatography system (FPLC), at a flow rate of1 ml min71. The protein was eluted using a linear gradientfrom 0 to 1 M NaCl. Nuclease A was eluted between 300and 400 mM sodium chloride, while nuclease B was elutedbetween 180 and 250 mM NaCl. Fractions were analysed bySDS–PAGE (17.5%). Fractions containing pure proteinwere pooled and concentrated using an Amicon concentratoron a Diaflo YM-10 membrane. Purified protein was found tobe 98% pure as judged by SDS–PAGE (data not shown).

Nuclease-activity assays

The specific activity of nuclease A and B was determinedusing heat-denatured salmon-sperm DNA by the method ofCuatrecassas et al. (1967) with the following changes. Reac-tions were performed at room temperature. The assays wereperformed in 1 ml reaction mixtures containing 25 mM Tris-HCl, pH 7.5, 10 mM CaCl2, and DNA at a concentration of40mg ml71. The enzyme concentration in the assay mixturewas approx. 0.02mg ml71, having been diluted in 0.1%DNase-free BSA. One unit of the enzymatic activity is definedas the amount of enzyme in milligrams causing a change of1.0 absorbance unit per min at 260 nm in a 1 cm cuvette.Reaction velocity was determined from the portions of therecorder trace, which typically remained linear for approx.0.5 min. One unit of specific activity is the absorbancychange per min per mg of protein. Protein concentrationswere determined by absorbance at 280nm (E1.0% at 280 nm=9.3 at neutral pH). The activity units do not take into accountthe 19-residue pro-peptide.

Circular dichroism (CD)

Equilibrium CD measurements were performed on an

automated Aviv 60DS spectrophotometer. The proteinswere in 10 mM sodium phosphate buffer, pH 7.5. Quartzcylindrical cells (Hellma) of path lengths ranging from 1 mmto 1 cm were used. Protein concentrations were not allowedto exceed 0.5 mg ml71 which corresponds to a maximumOD280 of approx. 0.5. Protein solutions and baselines werescanned at least three times and the scans averaged beforebaseline subtraction. Baseline scans were taken of buffersolutions without protein. The wavelengths ranged from 250to 200 nm for the far-UV, and between 250 and 330 nm forthe near-UV.

A temperature melt from 20 to 708C in 28C increments wasperformed on the two proteins, using the temperature mode ofthe machine. Proteins were equilibrated for 1 min per 8C, andan averaging time of 10 s was used in collecting data at eachtemperature. The melts were shown to be reversible both fromthe superimposability of scans that were performed on theprotein solutions before and after melting, and by the super-imposability of melts that were run backwards from 70 to208C. The van’t Hoff enthalpy (DHvan’tHoff) and the meltingpoint (Tm) were estimated by performing curve fitting onthe raw data of the change in ellipticity at 222 nm as a functionof temperature, as in Greenfield et al. (1994). The correlationcoefficient was 50.997. The van’t Hoff enthalpy was con-verted to free energy (DG) at 293 K.

Acknowledgements

This work was supported by a grant from the AmericanCancer Society (ACS grant MV-499Q). We would like tothank Donald Oliver of Wesleyan University for providing uswith the SecA temperature-sensitive strains. We also wishto thank Yoshikazu Nakamura of the University of Tokyo forproviding us with the signal peptidase temperature-sensitivestrains. We would also like to thank Ujwal Shinde for a criticalreading of this manuscript.

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the 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in...

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