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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 264, No. 28, Issue of October 5, pp. 16591-16597.1989 Printed in U.S.A.

Circular Dichroism Studies on Synthetic Signal Peptides Indicate @-Conformation as a Common Structural Feature in Highly Hydrophobic ]Environment*

(Received for publication, May 10, 1989)

G. Laxma IXeddy and R. NagarajS From the Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

The conformations of synthetic peptides corresponding to signal sequences of chicken lysozyme and Esch- erichia coli proteins alkaline phosphatase and lipoprotein (wild-type) and their ‘variants” with a charged amino acid in the hydlrophobic region, have been stud- ied by circular dichroism spectroscopy in trifluoroethanol and micelles of sodium dodecyl sulfate, Brij 36, and sodium deoxyclholate. In trifluoroethanol and aqueous mixtures of trifluoroethanol, the “wild-type” and variant signal sequences show similar conformational behavior. The wild-type signal peptides show increasing amounts of @-structure going from sodium dodecyl sulfate to deoxycholate micelles (i.e. increasing order of hydrophobicity). The variant signal sequences, however, are largely unordered in micelles. The absence of @-structure in variant signal sequences which do not initiate protein translocation across membranes, strongly suggests that the ability of signal sequences to adopt @-structure in a highly hydrophobic environment is important for function.

The molecular address that ensures targeting of secretory proteins to the endop1.asmic reticulum in eukaryotes and periplasm and outer membrane proteins to the inner membrane of Escherichia coli are peptide sequences 20-25 residues in length (1-4). These peptide segments, called signal sequences, occur transiently at the amino terminus of newly synthesized export proteins. After initiating translocation across membranes they are cleaved by membrane-bound signal peptidases (2). The primary structures of a large number of signal sequences have been determined (5). The only common feature that is clea.rly discernible is a positively charged region followed by a hydrophobic segment. In an attempt to delineate common structural features, the conformations of signal sequences have bleen examined by theoretical analysis (6) and experimental methods such as circular dichroism (7- 12). Theoretical analysis indicates the possibility of both a- helical and @-structure (6). Circular dichroism studies on E. coli X-receptor (8), Pho I{ gene product (12), “13 coat protein (lo), and parathyroid hormone (7) signal sequences have indicated predominantly a-helical conformation in trifluoroethanol and sodium doclecyl sulfate (SDS)’ micelles, whereas

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence should be addressed. The abbreviations used are: SDS, sodium dodecyl sulfate; Boc, t-

butyloxycarbonyl; DMF, dimethylformamide; HOBT, l-hydroxybenzotriazole; MeOH, methanol; TFE, trifluoroethanol; Z, benzyloxycar- bonyl; dansyl, 5-dimethylaminonaphthalene-l-sulfonyl.

the signal sequence of E. coli alkaline phosphatase (13) shows both a-helical and @-structure in these media. Thus a consen- sus conformational feature does not emerge from experimental studies either.

The hydrophobic region plays a critical role in the function of signal sequences. This is highlighted by extensive genetic studies in E. coli (2) and in vitro reconstitution experiments in eukaryotes (14). Reduction in the overall hydrophobicity by introduction of amino acids such as P-hydroxyleucine or reduction of the length of the hydrophobic stretch by introduction of charged amino acids renders signal sequences nonfunctional. That is, such mutant signals are unable to initiate translocation of proteins across membranes (2). While it is unlikely that such alterations would result in drastic structural alterations, they could conceivably affect the interaction of signal sequences with hydrophobic surfaces. Signal sequences, in the process of targeting of secretory proteins to membranes and initiating translocation of proteins across membranes encounter hydrophobic surfaces of different kinds, such as the signal recognition particle (15), the endo- plasmic reticulum, and inner membrane in E. coli.

In an effort to explore the conformational properties of wild-type signal sequences and “variants” which do not initiate translocation of proteins across membranes, in media of different hydrophobicities, we have carried out circular dichroism studies on synthetic peptides corresponding to the signal sequences of E. coli proteins, alkaline phosphatase and lipoprotein, and chicken lysozyme and their variants with charged amino acids in the hydrophobic regions (Fig. 1) in SDS, Brij 35, and sodium deoxycholate micelles. Extensive fluorescence studies have shown that there is an increase in hydrophobicity in the interior of micelles going from SDS to Brij 35 and sodium deoxycholate (16). We observe that going from SDS to sodium deoxycholate micelles (increasing order of hydrophobicity) the peptides corresponding to wild-type signal sequences show increasing amount of @-structure. The variant sequences with charged amino acids in the hydrophobic region are conformationally similar to wild-type sequences in media like TFE, MeOH, and aqueous mixtures of these solvents. However, going from SDS to deoxycholate micelles, the variants tend to be unordered rather than adopting any preferred conformation.

EXPERIMENTAL PROCEDURES

Amino acids were from Sigma. Protected amino acids were synthesized by established procedures. Merrifield resin (1% cross-linked), diisopropylethylamine, trifluoroacetic acid, dicyclohexylcarbodiim- ide, trifluoroethanol, SDS, and l-hydroxybenzotriazole were from Sigma.

Synthesis of Peptides-Peptide 1 corresponding to the signal sequence of chicken lysozyme was synthesized entirely by solution phase methods. The scheme for the synthesis is outlined in Fig. 2.

16591

16592 Conformation of Synthetic Signal Peptides in Micelles \ I

WJld-type signal sequences :

FIG. 1. Primary structures of synthetic signal peptides. Peptides 1- 3 correspond to signal sequences of chicken lysozyme and E. coli proteins, alkaline p h ~ p h a ~ s e , and lipoprotein, respectively (5). Peptides 4-6 correspond to variants of 1-3 with charged amino acids in the hydrophobic region.

Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(5zl~-Phe-Leu-Pre-Leu-Ala-Ala-Leu-Gly 1 1 5 10 15

Lys-Gln-Ser-Th+-Ile-Ala-Leu-Ala-Leu-I~eu-Pro-Leu-Leu-Phe-Thr-Pre-Val-Thr-Lys- 1 5 10 15

Ala-OCH3 2 20

Met-Lys-Ala-Thr-Lys-~eu-Val-Leu-Gly-Ala-Val-I3e-Leu-Gly-Thr-Thr-Leu-Leu-Ala- 1 5 IO 15

Gly-OCH3 2 20

\ # Variant signal sequences :

Lys-Leu-Leu-Ile-Ala-Leu-Val-Leu-Lys-Phe-Leu-Pro-Leu-Ala-A1a-Leu-Gly-OCH3 4 I 5 IO 15 -

I,ys-Gln-Ser-Thr-Ile-Ala-Leu-G~~~-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala-O~H~ 4 1 5 10 1 5

-

Lys-Ala-Thr-Lys-Leu-Val-Leu-Gly-Ala-Lys-Ile-Leu-Gly-Thr-T~r-Leu-Leu-Ala-Gly- 5 i o 15 I

OCH3 5

Couplings were mediated by DCC in CH2ClZ for dipeptides and DCC/ HOBT in DMF for longer peptides. Saponification was done to generate peptide acids. Peptide-free bases were obtained by deprotec- tion of the Boc group by HCl/tetrahydrofuran followed by neutralization with NazC03 solution and extraction with CHCla. Protected peptides were purified by column c h ~ m a t o ~ a p h y on silica gel. The desired peptides were eluted with varying proportions of MeOH/ CHCls. The fully protected peptide 1 was treated with trifluoroacetic acid, thioanisole, and metacresol (3.5 0.35, 0.35 v/v) to remove the protecting groups. The deprotected peptide was purified by partition c h r o m a ~ ~ a p h y on LH-20 as described (17). Peptide was hydrolyzed with trifluoroacetic acid/HCl (k1) for 48 h. After removing trifluoroacetic acid and HCl, the hydrolysates were reconstituted in 0.1 M NaHC03 and labeled with dansyl chloride. Analysis of the dansylated amino acids by high-pressure liquid chromato~aphy (Hewlett-Pack- ard 1090 instrument) on a Waters p-Bondapak (3.9 X 30 mm) Cls column with a mobile phase of 20% acetonitrile in an aqueous solution con~in ing 5 mM L-protine and ammonium acetate and 2.5 X M CuS04.5Hz0 (pH 7.0) (18) at a flow rate of 1 ml/min did not reveal the presence of any D-isomers of Phe, Ile, Lys, Leu, Ala. Hence this rules out racemization during the course of the synthesis of peptide.

Peptides 2, 5, and 6 were synthesized entirely by solid-phase methods by procedures described earlier (17, 19). The first amino acid (ie. COOH-terminal amino acid, Gly) was attached to the resin by the cesium salt procedure of Gisin (20). The extent of substitution was determined by the picric acid method (21). Substitution of 0.6 meq/g was used for the synthesis. One cycle of synthesis consisted of the following operation: 1) CHZCI, wash, 10 ml, 3 X 1 min; 2) 30% trifluoroacetic acid/CHzC12, 10 ml, 30 min; 3) CHXClz wash, 10 ml, 3 x 1 min; 4) CHzCls wash, 10 ml, 5 X 1 min; 5) 5% diisopropylethy1~- ine in CH2C12, 10 ml, 1 min (prewash); 6) 5% diisopropylethylamine in CH2Cl2, 10 min; 7) CH&& wash, 10 ml, 3 X 1 min; 8) Boc-amino acid in CHzClz (5 eq of initial substitution) 5 min; 9) DCC in CHZCls, 2 h; 10) 33% EtOH in CH2ClZ, 10 min; 11) repeat steps 9-11; 12) picric acid test: followed by either coupling or step 1. Peptides were cleaved off the resin by transesterification and the crude, protected peptides were purified by column chromato~aphy on sifica gel followed by partition chromatography on LH-20. Deprotected peptides were obtained by treatment with trifluoroacetic acid, thioanisole, metacresol (3.5, 0.35, 0.35, v/v) (22). Peptide 3 was synthesized by coupling the fragment Boe-Met-Lys(Z)-Ala-Thr-Lys(Z)-OH (synthesized by solution phase methods} to the peptide Boc-Leu-Val-Leu- Gly-Ala-Val-Ile-Leu-Gly-Thr-Thr-Leu-Leu-Ala-Gly bound to the resin as follows. Depmtection and neutralization of the resin-bound peptide was carried out as described above. Peptide Boc-Met-Lys(Z)-

Ala-Thr-Lys(Z)-OH in DMF was added followed by HOBT. After 10 min, DCC was added and the coupling was allowed to proceed for 24 h. The coupling was performed twice. Peptide 4 was obtained by coupling Boc-Lys(Boc)-Leu-Leu-Ile-Ala-OH (synthesized by solution phase methods) to Leu-Val-Leu-Lys(2)-Phe-Leu-Pro-Leu-Ala-Leu- Gly-methyl (synthesized by solid-phase methods and cleaved from resin by transesterification) in DMF with HOBT. Amino acid analysis of the purified peptides were performed on an LKB 4151 Alpha Plus amino acid analyzer after hydrolysis in 6 N HC1 at 100 "C in uacuo for 24 h and hydrolysis in trifluoroacetic acid/HCl (1:l) a t 100 "C in ucreua for 12 h. Hydrolysis with 6 N HCl yielded correct Thr values but slightly lower Ser values. Hydrolysis with trifluoroacetic acid/ HCl showed expected values for all amino acids except for Ser and Thr where >25% loss was observed. Nonleucine was used as an internal standard. The amino acid analyses are summarized in Table I.

Peptide I differs from the signal sequence of chicken lysozyme at position 2 where Lys has been used instead of Arg. Likewise in peptide 3, Thr at position 16 has been used instead of Ser. These changes are conservative and have been shown not to affect signal sequence function by genetic methods (2). These replacements were done primarily to facilitate synthesis. For peptide 4, Ala was intro- duced at position 5, as coupling yields were very low without Ala. Peptide 5 with Nitro-Phe instead of Phe was synthesized primarily for photochemical work.

Circulur ~ ~ h r o ~ ~ Studies-Circular dichroism spectra were re- corded on a Jobin-Yvon Dichrograph V spectropolarimeter in a 0.1- mm cell a t 25 "C. Mean residue ellipticities were calculated using the following equation:

1% = x A x 3300 x M

where A = observed dichroic absorbance, 1 = path length in centi- meters, C = concentration of peptide in grams/liter, and M = mean residue weight.

Peptides were weighed accurateIy (5 mg) and stock solutions were prepared in the appropriate solvent. Concentration of peptide was determined by quantitative amino acid analysis on an LKB 4151 Alpha Plus amino acid analyzer. For studies in micelles, samples were prepared as follows: an aliquot of peptide and the appropriate micelle forming compound in organic solvent were mixed and dried. The dried mixture was reconstituted in water with vortexing. In all cases, clear solutions were obtained. The spectra were fit with the reference spectra of polylysine (23). The mean residue ellipticity a t X can be

Conformation of Synthetic Signal Peptides in Micelles 16593

Met Lys Ser Leu Leu Ile Leu Val Leu Cys

Bot

Boc

3H H

, Phe Leu Pro Leu Ala Ala Leu Gly

FIG. 2. Outline of the strategy for the synthesis of peptide I by the solution phase method. Dipeptide couplings were mediated by DCC or DCC/HOBT. All peptide couplings were carried out in CH2CI2, longer peptides were coupled in DMF. Boc groups were removed by HCl/tetrahydrofuran and saponication was done in MeOH with 2 N NaOH.

TABLE I Arnim, acid analysis of synthetic s i g d peptides

Theoretical values are in parentheses.

ORrl

0821

OBzl

0B1l

OB21

0 8 2 1

OBzl

OB21

OBzl

OBzI

OBzi

OBz I

OH

Peptide Met Thx Ser Glu/Gln Pro Gly Ala Val Re Leu Phe Lys 1 0.95 0.73 1.00 1.13 1.82 0.95 0.80 7.20 0.91 1.00

(1) (1) (1) (1) (2) (1) (1) (7) (1) (1) 2".b 2.02 0.59

(3) 1.00 2.11

(1) 3.17 1.00 1.02 5.19 1.01 2.20

(1) (2) (3) (1) (1) (5) 3"

(1) 0.80 2.15

(2)

(1) (3) 2.93 3.18 2.00 0.88 5.14 2.20 (3)

4 (3) (2) (1) (5) (2)

1.12 1.00 3.00 1.00 0.89 7.12 1.00 1.95 (1) (1) (3) (1) (1) (7)

5* (1)

1.62 (2)

1.80 (2)

2.00 1.16 0.80 4.92 (2) (2) (1) (1) (5)

2.2

6b (2)

2.85 2.97 1.00 0.88 4.80 (3) (3) (1) (1) (5) (3)

3.14 2.15 (3)

Ser and Thr not correctad for loss during hydrolysis. Low values for Ser and Thr as h y ~ l y s i s was done with trifluomacetic acid/HC1 (1:l).

expressed as x (x) = fHx&) + fdB(x)fRxR(h). Where fH, fB, fR are sequence which is unable to initiate expo& of alkaline phos- fraction helical, & and random conformation; and XHS XB and XR phatase (24). Genetic studies have in&cat& that introduction corresponding values from reference CD spectra. The values for fH, fB, and fR were obtained by solving a series of simultaneous equations Of Lp in the middle Of the hy*ophobic re@on renders by the method of least squares using the constraint that fH + fB + fR sequences nonfunctional (25). Hence, peptides 4 and 6 were

MeOH, and aqueous mixtures of these solvents indicate that

Peptides 1-3 correspond to wild-type signal sequences. Pep- structure and 65% random conformation (11, 13). The CD tide 5 corresponds to mutant alkaline phosphatase signal spectra of peptides 4-6 which correspond to variants of signal

= 1. synthesized. The CD spectra of peptides 1 and 2 in TFE,

RESULTS these sequences have an a-helical content 30 and 15% 8-

16594 Conformation of Synthetic Signal Peptides in Micelles

sequences 1-3 with a charged amino acid in the hy~ophobic region in TFE are shown in Fig. 3. The spectra are characterized by negative bands -205 and -220 nm with crossover at 197 nm. The wild-type signal sequences 1 and 3 (shown in inset), as well as that of 2, also show double minima 205 and 220 nm. All these spectra indicate that the peptides have 30- 40% a-helix, 10-15% @-structure, and 50-60% random conformation. The CD spectra of peptides 4-6 and 1 and 3 in TFE/H,O (1:l) are shown in Fig. 4. Negative minima -205 and -220 nm are discernible for all the peptides. The spectra are not very different from those in TFE. Thus signal peptides

I

1-3 and their variants 4-6 show similar conformational behavior in TFE and TFE/H20.

The CD spectra of wild-type signal peptides 1 and 3 in SDS micelles are shown in Fig. 5. Peptide 1 shows a minimum at 212.5 nm with a crossover at 202 nm. The spectrum of 3 is characterized by a minima at 202 and 220 nm with crossover at 195 nm. The spectrum of 1 is characteristic of peptides in @-sheet conformation. Estimation of secondary structure parameters from the spectrum yields 80% ,&structure and 20% random conformation with no a-helical contribution. Esti- mation of secondary structure from the spectrum of peptide 3 yields 15% a-helical structure, 30% @-structure, and 55% random structure. In an earlier report (13) we had shown that peptide 2 also has both @-sheet and a-helical components in SDS micelles. The CD spectra of the variant signal sequences 4-6 in SDS micelles are shown in Fig. 6. Two minima at 206 and 220 nm are clearly discernible for all the peptides. Esti- mation of secondary structural parameters yields 15% a-helix, 40% @-structure, and 45% random conformation for 4 and 6,

FIG. 3. CD spectra of peptides in TFE. X, 4 (C = 0.2 mM); .-. , 5 (c = 0.1 mM); -, 6 (c = 0,065 mM). Inset: a, f (c = 0.09 mM); - - -, 3 (C = 0.089 mM).

FIG. 5. CD spectra of wild-type signal peptides in SDS micelles (16 mM). 0, l (C = 0.09 mM); - - -, 3 (C = 0.089 mM).

FIG. 4. CD spectra of peptides in TFE/HeO (1:l). X, 4 (C = 0.1 mM); 0-* , 5 (c 0.06 mM); -, 6 (c = 0.065 mM). bet: 0, 1 (c = 0.05 mM); - - -, 3 (C = 0.06 mM).

FIG. 6. CD spectra of variant signal peptides in SDS micelles (16 mM). X, 4 (c = 0.2 mM); *-*, 5 (0.13 mM); -, 6 (c = 0.089 mM).

Conformation of Synthetic Signal Peptides in Micelles 16595

FIG. 7. CD spectra of' wild-type signal peptides in Brij 35 micelles (0.13 mM). 0, 1 (C = 0.09 mM); A, 2 (C = 0.13 mM); _ " , 3 (C = 0.07 mM).

FIG. 8. CD spectra of variant signal peptides in Brij 35 micelles (0.13 mM). X, 4 (C = 0.2 mM); 0 - 0 , 5 (C = 0.13 mM); - , 6 (c = 0.89 mM).

FIG. 9. CD spectra of wild-type signal peptides in sodium deoxycholate micelles (7 mM). e, 1 (c = 0.09 mM); A, 2 (C = 0.013 mM); - - -, 3 (C = 0.07 mM).

and 15% a-helix, 25% @-structure, and 60% random conformation for 5.

The CD spectra of peptides 1-3 in Brij 35 micelles are shown in Fig. 7. The spectrum of peptide 2 indicates predominantly an unordered conformation. The spectra of peptides 1 and 3 show a single minima at 215 and 218 nm, respectively. The spectra, on analysis, yields 60% @-structure and 40% random structure with no a-helical structure. The CD spectra of peptides 4-6 in Brij 35 are presented in Fig. 8. The spectra

FIG. 10. CD spectra of variant signal peptides in sodium deoxycholate micelles (7 m~). X, 4 (C = 0.4 mM); .-*, 5 (c = 0.13 mM); "-, 6 (c = 0.089 mM).

TABLE I1 CD data for signal peptides

CD extrema Medium Peptide

X rsl,., x 10-3 X I S I ~ x 10-3

TFE

TFE/H20 1 3 4 5

SDS

Brij 35

6 1 3 4 5 6 1 2 3 4 5 6

Sodium deoxycholate I 2 3 4 5 6

nrn 205 205 205 205 207 205 204 205 203.5 205

203 206.5 205 207 200 200 198

201

nrn -17.2 222 -15.2 222 -19.7 222 -13.5 220 -18.4 220 -19.5 222 -16.5 222 -19.8 220 -11.0 222 -15.8 222

-13.5 222 -12.8 220 -12.5 220 -15.8 220

212.5

+5.0 215

+3.5 218

-11.2 222

214 214 222 215 215-220 215-225

-23.2 222

220-225

215-230

-10.4 -7.0

-10.0 -9.5

-15.0 -14.0 -8.0

-12.0 -5.6

-11.2 -11.4 -5.0 -9.0 -5.8

-12.0 -8.5 -4.5 -5.4 -3.6 -3.8 -1.6 -9.6 -7.9 -5.0 -8.0 -4.0 -2.0

indicates that these peptides do not adopt any preferred conformation in Brij 35, unlike peptides 1-3.

The spectra of peptides 1-3 in micelles of sodium deoxycholate is shown in Fig. 9. The peptides show a single minimum at 212 and 222 nm. However, the position of the minimum as well as the crossover point differ considerably for the three peptides. Estimation of secondary structure parameters indicab 80% @-structure and 20% random structure for 1 and 60% ,&-structure and 40% random structure for 2. The spectrum of peptide 3 does not resemble a typical @-sheet spectrum or a combination of a-helix and @-structure. However, a combination of 8-turn, especially type 11, and 8-sheet structure could conceivably result in a spectrum as depicted in Fig. 9. The spectra of peptides 4-6 in deoxycholate micelles are shown in Fig. 10. Peptides 5 and 6 clearly do not show any ordered conformation. However, the spectrum of 4 is very

16596 Confor~ution of ~ y n ~ ~ t ~ ~ Signal Peptides in ~ ~ e Z l e s

similar to that of the corresponding wild-type sequence indi- cating exclusively @-sheet conformation.

The CD spectra of peptides 1-6 were independent of peptide concentration in the range 0.05-0.25 mM in TFE, TFE/H20, and in micellar environment. The spectra were also independent of detergent concentration above CMC. The [@IM values at the peak extrema for peptides 1-6 in different media are summarized in Table 11.

DISCUSSION

The CD spectra of signal peptides 1-3 in TFE, MeOH, and aqueous mixtures of these solvents indicate the presence of a-helix and @-structure. The percentage of a-helix and @- structure is also variable among the peptides. However, predominantly a-helical conformation has been observed for other signal sequences in these solvents (7-10). Thus, in a medium like TFE, signal sequences exhibit considerable conformational flexibility. In micelles of SDS, almost exclusive @-sheet conformation is observed for the lysozyme signal sequence. The lipoprotein signal sequence exhibits both a- helix and @-sheet conformational features. A similar obser- vation was made with the signal sequence of alkaline phosphatase (13). On going to more hydrophobic micelles of Brij 35, exclusive &conformation is observed for both the signal sequences of lysozyme and lipoprotein whereas the alkaline phosphatase signal is more or less unordered. In micelles of deoxycholate all the three signal sequences 1-3 adopt exclusive ~-conformation. Since the CD spectra of peptides 1-3 are independent of concentration, it is unlikely that spectra characteristic of @-sheet conformation is observed due to peptide aggregation. A recent study on the signal sequence of the phoE gene product of E. coli indicates a marked preference for @-sheet conformation in micelles of Lubrol and lysoleci- thin (12). @-Conformation has also been observed for the signal sequence of E. coli X-receptor in the surface of lipid monolayers (26). While signal sequences of parathyroid hormone (7) and “13 coat protein (10) have been shown to adopt almost exclusive a-helical conformation in SDS micelles, studies in micelles of Brij or sodium deoxycholate do not exist for these peptides. While there are limitations in quantitating amounts of periodic structure in small peptides, like signal sequences based on reference spectra of large polymers (Greenfield and Fasman’s method), it is evident that signal sequences of varying primary structures tend to adopt almost exclusive @-st~cture in highly hydrophobic environments. Hence it is likely that the ability of signal sequences to adopt @-structure in highly hydrophobic environments may be functionally significant. Support for this argument comes from the study on peptides 4-6 which are variants of 1-3 with charged amino acids in the hydrophobic region. These peptides show conformational features similar to the wild-type peptides in TFE and aqueous mixtures of these solvents. However, in micellar environment, these peptides do not have a tendency to adopt @-struc~res. In fact, 5 and 6 are unordered in Brij 35 and deoxycholate micelles. Genetic studies have indicated that the “mutant” signal sequence 5 initiates translocation of alkaline phosphatase very inefficiently (24). Likewise, 6 also would be expected to be nonfunctional (25). Peptide 4, unlike 5 and 6, shows @-structure in deoxycholate micelles like the wild-type sequence 1-3. In this peptide, there are seven hydrophobic amino acids before and after Lys and is conceivable that such a sequence can initiate translocation of proteins across membranes.

In the intracellular sorting of proteins, signal sequences encounter and bind to a protein (such as signal recognition particle) which results in targeting of the ribosome-bound

nascent peptide chain of secretory proteins to the membrane site for translocation (15). Signal sequences then initiate translocation of secretory proteins across membranes. Essen- tially two schools of thought exist for explaining this step. One school argues for the interaction of signal sequence with a protein receptor which results in opening of an aqueous channel for translocation (15, 27). Another school postulates the direct partitioning of signal sequences into the hydrophobic interior of membranes as the first step in translocation (28-31). Therm~ynamic considerations argue for an a-helical conformation for signal sequences in order to span the lipid bilayer (30). We have shown that signal sequences can perturb the bilayer of lipid vesicles so as to cause fusion and also render them leaky to hydrophilic molecules like carboxy- fluorescein (31). We have postulated that signal sequences thus cause “local defects” in the lipid bilayer which may be responsible for protein translocation either directly or through rearranged protein channels formed as a result of lipid per- turbation. For this type of initiation of translocation, an a- helical conformation is not mandatory for signal sequences. Our results indicate that signal sequences adopt exclusively @-structure in highly hydrophobic environment. Other hydrophobic proteins such as “13 coat protein (32) and a-toxin (33) have been shown to adopt &structures in hydrophobic environment. The absence of @-structure in hydrophobic environment in our synthetic “mutant” signal sequences which do not initiate protein transport across membrane strongIy suggests that the ability of signal sequences to adopt 8- structure in hydrophobic environment is important for function.

A c ~ ~ ~ ~ ~ ~ n t s - W e thank V. Dhople for help in amino acid analysis, E. Bikshapathy for synthesizing one of the peptides, and M. V. Jagannadham for HPLC analysis of dansyl-amino acids. We are grateful to Prof. D. Balasubramanian for criticism and discussions.

1.

2. 3. 4.

5. 6. 7.

8.

9.

10.

11.

12.

13. 14.

15. 16.

17,

18.

19.

20. 21.

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circular dichroism studies on synthetic signal peptides ... · pdf filebutyloxycarbonyl; dmf,...

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