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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 correspond- ing to signal sequences of chicken lysozyme and Esch- erichia coli proteins alkaline phosphatase and lipopro- tein (wild-type) and their variants with a charged amino acid in the hydlrophobic region, have been stud- ied by circular dichroism spectroscopy in trifluoroeth- anol 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 conforma- tional behavior. The wild-type signal peptides show increasing amounts of @-structure going from sodium dodecyl sulfate to deoxycholate micelles (i.e. increas- ing order of hydrophobicity). The variant signal se- quences, however, are largely unordered in micelles. The absence of @-structure in variant signal sequences which do not initiate protein translocation across mem- branes, strongly suggests that the ability of signal se- quences 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 mem- brane of Escherichia coli are peptide sequences 20-25 residues in length (1-4). These peptide segments, called signal se- quences, occur transiently at the amino terminus of newly synthesized export proteins. After initiating translocation across membranes they are cleaved by membrane-bound sig- nal peptidases (2). The primary structures of a large number of signal sequences have been determined (5). The only com- mon 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 trifluoro- ethanol 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-hydroxyben- zotriazole; 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 experimen- tal 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 intro- duction of charged amino acids renders signal sequences non- functional. 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 struc- tural alterations, they could conceivably affect the interaction of signal sequences with hydrophobic surfaces. Signal se- quences, 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 initi- ate translocation of proteins across membranes, in media of different hydrophobicities, we have carried out circular di- chroism 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 hydropho- bic 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 synthe- sized 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 se- quence 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 neutrali- zation 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 trifluo- roacetic 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

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