CHAPTER - IV

CONFORMATIONAL STUDIES ON SIGNAL PEPTIDES

59

4.1 Introduction

An examination cd the primary structure of eukarY0tic and pr0karY0tic

signal sequences indicates the presence 0 E a p0sitively charged amin0-terrninus

f01l0wed by a ce-ntiguous stretch 0f 8-12 hydre-phe-bic amine- acid residues

(Watson, 1981J.). However, primary structure horne-10gy is absent in these se-

quences. Recent studies have indicated that the specific interactie-n e-f signal

sequences with c0mpC'nents C'f the expNt machinery, such as the "signal rec0g-

niti0n particle" (Walter and Ble-bel, 1981 a), is n0t sequence specific (Walter

and Ble-bel, 1981b, St0ffel et al., 1981, Anders0n ~ al., 1982, Muller et al.,

1982). The distributie-n of charged and hydrophobic amine- acids "may be e-ne

of the comme-n recognition elements in signal sequences. The specific recog-

niti0n 0f signal sequences e-f varying lengths and primary structure may als0

arise due to sec0ndary structural features. Applicati0n e-f the predictive rules

0f Ch0u and Fasman to signal sequences indicates that be-th a-and S -C0nfN-

matie-ns are probable (Austen, 1979). In an effNt te- find e-ut the conformati0nal

preferences of signal sequences in varie-us envir0nments, ce-nformati0nal studies

were carried 0ut e-n synthetic pep tides correspe-nding to the signal sequences

0f £.. coli proteins, lipopwtein (Jn0uye et aI., 1977) and its variants and frag-

ments, and alkaline ph0sphatase (Inouye ~ aI., 1982) by circular dichre-ism

spectroscopy.

4.2 Methods and Materials

Peptides used for CD-studies are presented in Fig. lJ..1.

4.2.1 Synthesis e-f peptides

Peptides l-2. were synthesized as described in chapter III.

Peptides I, ~, 2, &. and 2. differ frC'm their corresp0nding~. ce-li liP0-

pr0tein signal sequence, peptide l, at p0sitiC'n -6, .where Ser has been replaced

FIG. 4.1. Primary structure of peptides used for CD studies.

Peptides 1. E. coli lipoprotein signal sequence, l.

A variant of 1 with a Thr at position -6 instead

of Ser, 2. £. coli alkaline phosphatase signal sequence,

It. A variant of ! with Thr at position -6 and Lys

at position -10 instead of Ser and Val respectively,

5 - 2.. Frgments corresponding to various regions

of peptide! and l.

Peptides used for CD studies

1.

2.

3.

4.

5.

6.

7.

8.

9.

Met-L ys- Ala- Thr-Ly s-Leu-Val-Leu-Gly-Ala-Val-Ile-Leu-Gly-Ser- Thr-Leu-

Leu-Ala-Gly-OCH 3

Met-Lys- Ala - Thr-L ys-Leu-V al-Leu-Gly-Ala-Val-Ile-Leu-Gly- Thr-1l1r-Leu-

Leu-Ala-Gly-OCH 3·

L Y s-G 1 n-Ser- Thr- Ile- Ala-Leu-Ala-Leu-Leu-Pr0-Leu-Leu-Phe- Thr-Pr0-Val-

11lr-Lys-Ala-OCH3

L ys- Ala- Thr-L ys-Leu-Val-Leu-G ly-Ala-Lys-Ile-Leu-Gly- Thr- Thr-Leu-Leu-

Ala-Gly-OH

Boc- Leu- Val-Leu-Gly-Ala-Val-Ile-Leu-Gly-1l1r- Thr-Leu-Leu-Ala-Gly-OCH 3.

B0c-Gly-Ala-Val-Ile-Leu-Gly- Thr- Thr-Leu-Leu-Ala-Gly-OCH3

B0c-Leu-Val-Leu-Gly-Ala-Val-Ile-Leu-OCH3

B0c- Met-Lys-Ala- Thr-Lys-Leu-Val-Leu-OCH 3

B0c-Gly-1l1r- Thr-Leu-Leu-Ala-Gly-OCH3

60

by Thr. Peptide !!.- also differs in not having the amino-terminal Met, and at

position -10, where Val is replaced by Lys (a positive charge in the centre

of the hydrophobic core). The replacement of Ser by "Ihr at position -6 was

done for synthetic reasons, and this change is known in the signal to be conse-

vative, functioning normally at 37°C (Vlasuk et a1., 1984).

4.2.2 Preparation of samples for CD studies

Stock solutions were made either in methanol or trifluoroethanol,

depending upon the solubility of the pep tides. (Peptides.l and 1 were insoluble

in methanol). An aliquot of each peptide stock solution was sUbjected to quanti-

tative amino acid analysis as described in chapter II.

The samples for CD studies were prepared as follows: Appropriate

amounts of peptides were drawn from their respective stock solutions into

different tubes. For studies in micelles, a known amount of detergent in metha-

nol was added and the samples were dried in a Savant Speed - Vac-centrifuge.

In the case of micelles, the dried samples were reconstituted in water, and

in the other cases, in the appropriate solvent.

4.2.3 CD studies

CD-studies were performed on a Jobin-Yvon Dichrograph V spectro-

polarimeter in 0.1 cm cells at 25°C. Molar or mean residue ellipticities were

calculated using the equation:

[e J M =

D. A x 3300 x M c x 1

where A = observed dichroic absorbance, 1 = pathJength in cm, C = concent-

ration of peptide in gms/litre and M = Molecular weight or mean residue weight.

4.2.4

61

Method of secondary structure analysis from CD spectra

(Yang et~, 1986)

The experimental CD spectrum of a protein at each wavelength

can be expressed as:

n X (A) = I fiX i (A )

I -

Where X( A ) is the mean residue ellipticity at A ; fi is the fraction

of the ith conformation and Xi (A ) is the corresponding reference CD. The

reference CD spectra used here are poly-Lys, which exists in only one confor-

mation at a particular pH (Greenfield and Fasman, 1969). Then the fils are

solved from a series of simultaneous equations by a least square method, using

the constraint that the sum of the fractions of individual secondary structures

equals one.

4.2.5 CD of peptides and proteins

The main transitions characteristic of the peptide bond are the amide

* n- II * and II 11 • Depending on whether the solvent is strongly hydrogen * bonding or not, the n- 11 transition lies near 210 nm or 230 nm, respectively.

* The 11 - II transition, which is much stronger in magnitude, lies at lower

wa velength, 200 nm. These two transitions of the peptide group vary depending

on the conformation of the peptide backbone and generate the CD spectrum

characteristic of that particular conformation.

, a -helix

CD spectrum of a -helix is characterized by the presence of a negative

* band at 222 nm corresponding to n- H transition (Schellman and Oriel, 1962),

62

Woody and Tinoco, 1967), a negative band at 208 nm and a positive band at

192 nm due to exciton splitting of II - II * transition (Mandel and Holzwarth,

1972) and another positive shoulder at 175 nm due to n- a * transition (Johnson

and Tinoco, 1972).

The n- Jl * transition and thus the CD band at 222 nm, is relatively

insensitive to chain length (Woody and Tinoco, 1967, Madison and Schell man,

1972) and remains negative from the dimer upto the infinite helix, with a

gradual increase in magnitude as the chain length increases. The II --II * transition

at 210 nm is strongly chain length dependent, and is absent in shorter helices

less than 10 residues (Woody, 1985). FN the H -][ * transition at ~ 190 nm, a

positive band is present for all chain lengths, but its magnitude increases mar-

kedly with chain length.

S -sheet

The characteristic features of a S -sheet CD spectrum is a negative

band at ~ 216 nm due t(l n- it * transiti(ln, a p(lsitive band between 195 nm

and 200 nm due t(l II -II * transiti(ln and a negative band near 175 nm, p(lssibly

due t(l n- a * transiti(ln (Brahmset ~, 1977). TIle CD (If B -sheets is much

ll'1(lre variable than that (If a -helix, bdh in magnitude and p(lsition (If the

bands. This variability results fr(lm much br(lader range (If S -sheets available

c(lmpared t(l a -helices.

Relatively smaller changes are predicted in i3 -sheet CD with increasing

chain length N sheet width (W(l(ldy, 1969, Madis(ln and Schellman, 1972). The

n- 11* transiti(ln i.e., ~ 216 nm negative band, increases in magnitude with chain

length but decreases with increasing sheet width. The II - II * regi(l n is n(lt very sensitive t(l chain length N sheet width in C(lntrast t(l the cv -helix.

63

8 -turns

The CD spectrum 0f 13 -tur ns, type J a nd type II resemble the 8 -sheet

CO spectrum, but with red shifted weak n- J( * CD between 220 nm and 230

nm (W00dy, 1974), and a p0sltive It - II * band between 200 nm and 210 nm.

In additi0n a str0ng negative band is predicted between 180 nm and 190 nm.

TI1is CD pattern is referred to as class B CD spectrum, while the standard

I; -sheet CD spectrum is turned class A. An0ther, class C CD spectrum which

is ll-helix like is predicted fN type II'" turn. H0wever, a br0ad range 0f c0nfN-

mati0ns are described as B -turns, S0me 0f which may give rise to CD patterns

quite different from the 0thers.

Rand(lrn c(lil (lr uO(lrdered structure

The rand0m coil N unNdered peptides have very weak and highly

variable CD spectra. Generally, there is an intense negative band near ':O! 200

nm due to i! -Ji * transiti0n and a weak 220 nm n- II.* band which may be either

p0sitive N negative~

4.2.6 Micelles

T0 study the c0nfNmation 0f the signal sequences in membrane mime-

tic media, micelles 0f SOS, Brij 35 and Na-cholate N Na-OOC were used.

The pr0perties of individual types 0f micelles are described in Table 4.1. The

hydr0pl10bicity in the interiN 0f micelles 0f SOS. SOS (NaCO, Brij 35 and Na-

Ch01ate N Na-OOC increases in that Nder (Sh0bha, 1986).

4.3 Results

TI1e CD data 0f peptides l to 2. is summarized in Table 4.2. Sec0ndary structure parameters estimated fr0m the spectra, calculated as described in

materials and meth0ds are presented in Table 4.3. after page 48.

TABLE 4.1. Structure and properties of the micelles used in

CD studies.

S I.No. Name Abbreviation

Sodiumdodecyl sui phote SOS

2 I Polyoxyethylene (23) laurylether Brij - 35

3 I Sodium deoxycholate NaOOC

4 I Sodium cholate Nacholate

Structure

- + CH3(CH2)1I-0- S03Na

C'2 H25{ OCH2 CH 2 )23 OH

HO

HO

CMC in mM

8·0

0·065

3·5

4·0

64

Peptide!

TIle spectra 0f peptide 1 corresp0nding to E... coli lipoprotein signal

sequence in neat lFE and lFE/H2

0 mixture are sh0wn in Fig. 4.2 • The spec-

trum of the peptide in lFE is charactf'rized by a cr0SS over at 198 nm, negative

bands 0f 206 nm and 222 nm. In lFE/H2

0 (2'. \ ) the cross 0ver is shifted

to 197 nm, \vith a small decrease in the intensities of both 204 nm,

ard 222 rm regative bar:ds. Secondary structure analysis shows ~ 30% a -helix, ~

20% 8 -structure and ~ 50% unordered in lFE and lFE/H 20. The CO spectra

0f the peptide in micelles of 50S and Brij 35 are shown in Fig. 4.3. In 50S,

the spectrum sh0ws a cross over at 198 nm with distinct 206 nm and 220 nm

negative bands. In Brij 35, the cr0SS 0ver is at 207 nm, and a minimum at

217 nm, b0th characteristic 0f a 8 -structure. Secondary structure analysis

of the CD spectrum of the peptide In 50S sh0ws an increase in 8 -structure

to 40% and a corresp0nding decrease in helical content. On the 0ther hand

in Brij 35, 8 -structure of ~ 70%, with no helical content, is observed.

Thus peptide 1 adopts both a and 8 -structure in lFE and lFE/H 20 mixture. In ani0nic micelles like SOS, there is an increase in 8 -structure with

a corresponding decrease in helical c0ntent. In more hydr0phobic micelles

like Brij 35, the peptide ad0pts as much as 70% p -structure, with n0 helical

c0ntent.

Peptide 2

The CD spectra 0f peptide l, which differs from peptide 1 at p0siti0n

-6, where Ser is replaced by Thr, in neat lFE and the lFE/H 20 are shown

in Fig. 4.4. The CD spectrum in lFE is characterized by a cr0SS 0ver at 196 nm

with negative bands at 203 nm and 222 nm. In lFE/H20, the spectrum sh0ws

a cr0SS ewer at "'" 196 nm, and negative bands at 204 nm and 222 nm. Sec0ndary

FIG. 4.2. CD spectra of peptide 1- in TFE (--) and TFE/H20, 2: 1 (- - - -). Concentration of the peptide in TFE

== 0.063 mg/ml and TFE/H20, 2:1 == 0.042 mg/ml.

A (nm) 20'0 __ 2_0~0 ____ 2~10 _____ 2 __ 2_0 ____ 2~3~0 _____ 2_4_0 _____ 2_50

15·0

0 10·0

E "C

C\I 5·0 E (.) . Cl CIJ

"C 0·0 ro \ '0 ~

X

::E -5,0

11

FIG. 4.3. CO spectra of peptide 1. in SOS (--) and Brij 35(- - - -). Concentration of peptide in 50S = 0.063 -

mg/ml and Brij 35 = 0.025 mg/ml.

Ol Q)

-0 ro 10

)(

A. (nm) 200 210 220 230 240 250

20'0~~------~--------------~----~------~

15·0

10·0

" , 5·0 , ,

\ \

O.Or-r-----\7---------------------~~--~ \ , -5,0

, ,,-' , ,," , ",," '---~ -100 ,..,

FIG. 4.4. cn spectra of peptide ~ in TFE (--) and TFE/H20,

2: 1 (- - - -). Concentration of the peptide in TFE

= 0.178 mg/ml and TFE/H20 = 0.119 mg/m1.

A (nm) 200 210 220 230 240 250

20'O--~------~------~------~------~----~

15-0

0 10-0 e

-C

Ne 5-0 0 . 0\ CIJ -c 0-0

ro '0

x ~

-5-0

n

65

structure analysis shows almost equal amounts of a -helix and S -structure with

'" 55% unordered conformation in both TFE and TFE/H20. The CD spectra

of the peptide in SDS, Brij 35 and Na-DOC micelles are presented In Fig.

4.5. In SDS micelles, the spectrum IS qualitatively similar to that of in TFE,

except for a small decrease in the intensities of the bands, which by secondary

structure analysis shows 33% S -structure with very little helical content and

'" 55% unordered conformation. In Brij 35, the cross over is at 206 nm, with

a positive band at 198 nm and a negative band at 218 nm. In NaDOC, the cross

over IS at 208 nm and the negative bard at 222 nm. These spectra are characteri-

stic of peptides adopting a S -structure. Secondary structure analysis indicates

65-70% S-structure with no helical content.

Peptide ..£ differs from peptide 1. at position -6, where Ser residue IS replaced by a Thr residue. Genetic studies have indicated that this replace-

ment in peptide ..£ does not alter the function of the signal sequence (Vlasuk

~ ~., 1984). Conformational studies of peptide 2 in various environments like

TFE, TFE/H2

0 and anionic micelles like SDS and more hydrophobic micelles

like Brij 35 and NaDOC, also indicates that Ser to_lhr change does not result

in any drastic conformational change.

Peptide 1

CD spectra of peptide 1, corresponding to the !:.. coli alkaline phos-phatase signal sequence, in TFE and TFE/H20 are shown in Fig. 4.6. The CD

spectrum in TFE is characterized by a 197 nm cross over, one negative band

at 207 nm, and the other broad negative band spanning 218-222 nm. In TFE/H20

the cross over is shi [ted to below 195 nrn and the negative band to 203 nm,

while the other negative band remains at 222 nrn, with a slight decrease 111

the intensi ties of the bands indicating morE' unordered structure compared to

FIG. 4.5. CD spectra of peptide ~ in SOS (--) Brij 35

(- - - -) and NaDOC ( ..••.. ). Concentration of the

peptide in SDS = 0.178 mg/ml, Brij 35 = 0.143 mg/ml

and NaDOC = 0.143 mg/ml.

20·0

15·0

Ie; 10.0 E -0

o . 0" QJ

5'0

A (nm) 200 210 220 230 240 250

". t •••

'. ""..., .... , -.. -0

ro 10

• , .... 0·0n-----~~~ .. ----------------------~~~~

x :i

r-'I CD u

-5,0

-10'0

-15,0

. , t •• ,". ~

,.:.... ('t"-~ ...... .... ~~ ....... ...:,. ........... -

-20·0~----------~----------------~--------

FIG. 4.6. CD spectra of peptide 1. in TFE (--) TFE/H20-

2:1 (- - - -). Concentration of the peptide in TFE

= 0.2 mg/ml and TFE/H20 = 0.2 mg/ml.

5·0

2·5

0·0

0 -2,5 E -

"'0 -5,0 (\Ie

-7,5 0

0\ -10'0 Q) "'0

ro -12,5 '0

x -15,0 :E -17,5 1'1

66

TF E. Estimation of secondary structure parameters as described in materials

and methods yields 50% B -structure with no (i -helical structure. In MeOH and

MeOH/H2

0 mixtures (Fig. 4.7)~ negative bands 204 and 220 nm are observed.

With increasing concentration of H20, the 220 nm band remains unchanged,

whereas there is a slight increase in intensity as well as a blue shift of the

204 nm band. Estimation of secondary structural parameters yield a small amount

of helical conformation and B -structure of~40 %. With increasing proportion

of H20, the percentage of random structure increases. On the other hand, com-

parison of the spectra of peptide 3 in TFE, MeOH and their water mixtures

with the calculated CD spectra of Greenfield and Fasman (Greenfield and

Fasman, 1969) have indicated presence of 10-15% a -helix and 30-40% B -sheet.

The CD spectra of peptide l in SDS and SDS/NaCI are shown In Fig. 4.8. In SOS and SDS/NaCI, the spectra are characterized by cross over at 196

nm and 197 nrn, negative bands at 204 nm arid 206 nm, and broad negative bands

at 220-222 nm and 218-222 nm respectively. Apart from the overall red shift

of the spectrum in SDS/NaCI, there is a decrease in intensity of the extrema

compared to the one in SDS. The only difference in the micelles ofSOS and

SDS/NaCI is that the former forms spherial micelles and the latter forms egg

shaped micelles accompanied by an increase in hydrophobicity in the interior

of the micelles (Shobha, 1987). The CO spectra of peptide l in Brij 35 and

NaOOC are shown in Fig. 4.9. The CD spectrum of peptide 1. in Brij 35 shows

a more random structure with very little ordered structure. In NaDOC, the

spectrum shows a cross over at 197 nm and characteristic B -structure band

at 214 nm. Secondary structure analysis shows 50% B -structure without any

helical content.

Peptide 3 shows predominantly !3 -structure In various environments

FIG. 4.7. CD spectra of peptide l in MeOH (--) , MeOH/H2

0

- 3:1 (- - - -) and MeOH/H2

0 - 1: 1 (- •••• -). Concent-

ration of the peptide = 0.2 rng/rnl.

I o E

-0.

· 01 Q)

-0 ro 10

5·0r---------------------------------------~

2·5

0·0~r_----------------------------_=~~~

-2,5

-5,0

-7,5

\ -10,0 ". /

\ .I x -12,5 :!E

.. / ... " .... ..., -15,0

FIG. 4.&. CD spectra of peptide 1 in 5D5 (--) and 505/0.51\1\ NaCl (- - - -). Concentration of the peptide = 0'.138

mg/ml.

0 E

-0

(\IE 0

Ol Q)

-0 .. J'{)

10

)(

:E ...., (D L-J

200

15·0

10·0

5·0

0·0 \ \

\ , -5,0

,

-10,0

-15'0

210

" ..... _--' ",

A (nm) 220

"".-----

230 240 250

-20 0 '---------------------------

FIG. 4.9. cn spectra of peptide l in Brij 15 (--) and NaOOC

( ........ ). Concentration of the peptide = 0.138 mg/ml.

I o E

"'C

o

x

A (nm) 200 210 220 230 240 250

20·0--~------~-------------r------~----~

15·0

10·0

, • . ,

O·O~'~------------------------------~~~ , , , , , , , , , ,

, " " " "

:E -5-0 11

'" , ..... '"

,,-, "

"

, ' " ' .....

(J) " L-I

-10,0

-15-0

-20·0------~--------------------------------

67

like TFE, MeOH and micelles 0f SDS, SDS/NaCl, and Na-DOC. As in the case

of peptide 1 and I, there is a decrease in S -structure with a corresponding

increase in unordered conformation for the peptide l, as the polarity of TFE

and MeOH is increased by the addition of water. However, unlike in the case

of peptides 1 and I, there is no significant increase in S -structure with increas-ing hydrophobicity of micelles. Peptide l differs from 1 and I in that it has one Lys residue at the carboxy-terminus and two Pro residues, one in the centre

of the sequence and the other towards the carboxy-terminus. The amount of

S -structure is less in peptide l as compared to 1 and I due to the presence

of two Pro residues which are known to be helix and S -sheet breakers. It is

also conceivable that positive charges present at either end of the peptide

prevent intimate association with the micelles of Brij 35.

Peptide .9:

CD spectra of peptide ~, which differs from E. coli lipoprotein

signal sequence in having a Lys (positive charge) at position -la, instead of

Val, in TFE and TFE/H20 are shown in Fig.4.1O. The CD spectrum in TFE is

characterized by a cross over at 199 nm, and negative bands at 207 nm and

'" 222 nm. In TFE/H20, the cross over is at 198 nm, the shorter wavelength nega-

tive band at 205 nm and the negative band at 222 nm. However, there is a

decrease in the intensity of the bands in TFE/H 20 compared to the spectrum

in TFE. Secondary structure parameters obtained from these spectra indicate

33% helix and 25% s-structure in TFE, and 23% helix and 30% s-structure

in TFE/H20, the unordered being '" 45% in both cases. The CD spectra of this

peptide in MeOH and MeOH/H20 are shown in Fig. 4.11. The spectra in MeOH

and MeOH/H20 are characterized by a cross over at '" 197 nm and below 195

nm, a shorter wavelength negative band at 205 nm and 200 nm. and the longer

wavelength negative band at 222 nm and 218-222 nm respectively. Secondary

FIG. 4.10. CD spectra of peptide!!.. in TFE (--) and TFE/H20

- 2: 1 (- - - -). Concentration of the peptide in TFE .

= 0.196 mg/ml and TFE/H20 = 0.130 mg/ml.

A (nm) 200 210 220 230 240 250

20'0~~----~~----~----~------~----~

15'0

1- 10·0 0 E \ -0 \

(\IE \ 5·0 \ 0 01

\ Q) \ -0 0·0 ro

10 \ \ x ~ ::E

-5,0

M

FIG. 4.11. CD spectra of peptide 4 in MeOH (--) and MeOH/

H20, 2:1 (- - - -). Concentration of the peptide

in 'v\eOH = 0.145 mg/rnl and in MeOH/H2

0 =·0.098

mg/ml.

A (nm) 200 210 220 230 240 250

20'0~~------~------~------------~------~

15·0

1-10·0 0

E "'C

(\IE 5·0 0 . Ol Q)

"'C 0·0 ro '0 ---x ,,---

:E -5,0 /

" rt /

68

structure analysis shows that the peptide has '" 50% 8 -structure with very

little or no helical content in MeOH and MeOH/H20. The CO spectra in SOS,

Brij 35 and Na-cholate are shown in Fig.t+.l2. The spectrum in SOS is character-

ized by a 199 nm cross over, and negative bands at 707 nm and 220 nm. Second-

ary structure analysis indicates 23% helix and 35% 8 -structure. In Brij 35 and

Na-cholate, the spectra are characteristic of unordered conformation.

Introduction of a charged residue in the hydrophobic core is known

to render the signal sequence non functional (Michaelis and Beckwith, 1982).

Peptide !± corresponding to~. coli lipoprotein signal sequence, with a positive

charge (Lys) in the centre of the hydrophobic core, unlike peptides 1. and l

shows almost equal amounts of a and 8 -structures in TFE, MeOH and SOS

micelles. Also, unlike in the case .of peptides J and 2 increasing polarity

of TFE and MeOH by the addition of water causes a decrease in a -helix, with

a corresponding increase in 8-structure. Peptide !± is characterized by a largely

unordered structure in micelles of Brij 35 and Na-cholate.

Secondary structue prediction methods (Chou and Fasman, 1978) also

indicate that peptides 1. to !± have equal probability of adopting both a -helix

and 8-sheet. The average helix parameter « Pa > ), and average 8-structure

parameter « P 8 > ), for peptides 1. and l are 1.08 and 1.13, and 1.09 and 1.12

respectively. For peptide 1. « Pa» is 1.15 for residues Ser(-18) to Leu(-Il} and

« P8 » 1.12 for residues GIn (-19) to Leu(-ll) and 1.18 for residues Leu(-9)

to Thr (-6). For peptide !±«Pa » is 1.08 ancl«PS» is 1.13.

Pep tides ~ and ~

The CD spectra of pep tides .2.. and .§., corresponding to the carboxy

terminal 15 and 12 residues respectively, of ~. coli lipoprotein signal sequence

in TFE are shown in Fig. 4.13. The CD spectrum of peptide .2.. shows a negative

band at 205 nm and another negative band at 220 nm, while that of peptide

FIG. 4.12. CD spectra of peptide!:±. in 5DS (--), Birj 35 (-

-) and Na cholate ( ••.••. ). Concentration of the

peptide = 0.196 mg/rnl.

1-0 E

"'C

NE 0

C)

CU "'C

ro 10

x ~ ....,

CD L..I

A (nm) 200 210 220 230 240 250

20'0~--------------------------------------

15'0

10·0

5'0

0·0

.' . -5,0

-10,0

-15,0

....... -........ -------....... . ... :,;:.:. ---- .-•..... . ~.-r:-: ....... ....... ~ .' r .. . ' /

I

I I

I

/

-20·0--------------------------------------~

FIG. 4.13. CD spectra of peptide 1. (--) and ~ (- - - -) in TFE.

Concentration of peptide 1. = 0.64- mg/ml and peptide

6 = 0.715 mg/ml.

A(nm) 190 200 210 220 230 240 250

O·O----~----~--~----~----r---~~ .,,-

- 2·0

1 (5 -4·0

E

(\IE - 6·0 o Ol QJ-S·O

-C ¢

...

'a -10·0 x

:E CD -12·0 L..J

-14·0

\ " \ --\ / , ,I \. / -

I I

I I

I . I

I I

I

/

I I

I , " I

I

FIG. 4.14. CD spectra of peptides Z (--), 8 (- - - -) and 9 (_ .••• -) in TFE. Concentration of peptide Z = 0.45 mg/ml, peptide ~ = 0.5 mg/ml and peptide 2. = 0.425 mg/ml.

A(nm) 190 200 210 220 230 240 250

o·o----~----------~----~--~~~~~

-1,0 \

o \~ E ~. -2,0 -0

C\J

E -3,0 (,)

01 Q)

-0 -4,0 v 10

>< -5,0 ~

J1

TABLE 4.2. CD data concermng crossover points and CD extrema

of peptides 1 to 2..

Pep--tides

1 -

2

3

Solvent

lFE lFE/H

20 (2: 1)

SOS Brij 35

lFE lFE/H

20 (2: 1)

SOS Brij 35 Na-OOC

lFE lFE/H

20 (1: 1)

MeOH MeOH/H

20 (3:1)

MeOH/H2

0 0:1) SOS SOS/0.5M NaCl Brij 35 Na-!.}OC CHAPS

CrOSS0ver ).. (nm)

19&.0 197.5 19&.0

196.0 196.5 196.0 206.0 20&.0

197.0

197.5 195.0

196.5 197.5

197.0 200.0

).. (nm)

19&.0

CO extrema

[8 JIv1 x 10-3 ).. (nm) [8]~X10-3 )..(nm} [8 L x 10-3

-20.00 222.0 -14.24 204.0 -16.96 222.0 -11.52 206.0 -12.4& 220.0 -9.12

217.0 -5.92

203.0 -16.00 222.0 -7.20 204.0 -16.64 222.0 -&.4& 203.0 -13.44 222.0 -6.24

3.52 21 &.0 -5.2& 222.0 -5.12

207.0 -6.30 218-222 -3.10 207.0 -6.30 222.0 -2.90 205.0 -9.2 222.0 -3.70 202.5 -10.2 220-224 -3.40 200.0 -13.4 222.0 -3.70 204.0 -11.&4 220-224 -3.40 204.0 -5.60 21 &-222 -3.52 200.0 -16.&0 222-226 -3.6&

214.0 -7.&4 206.0 -7.68 222.0 -3.2&

(C0 ntd •••• /- •. )

Pep-tides

1+

5

6

7

8

9

Sol vent

1FE 1FE/H

20 (2:1)

MeOH MeOH/H?O (2: 1) 50S ~ Br ij 35 Na-cholate

1FE

1FE

1FE

1FE

1FE

Crossover A. (nm)

199.0 198.0 197.5

199.0

A. (nm) [eJM

x 10-3

CD extrema

A. (nm)

207.0 205.0 206.0 200.0 207.0

205.0

209.0

203.0

203.0

203.0

[6 J\t\ x 10-3 A. (nm) [8 ~\t\ x 10-3

-18.1+0 222.0 -11+.1+0 -15.68 222.0 -10.88 -10.16 222.0 -6.72

-7.68 ~~--, ~ LLu.'v -3.20 -15.81+ 220.0 -12.00

220.0 -1.60 220.0 -2.00

-9.90 220.0 -5.S

-12.30 220.0 - 0.50

-2.3 220.0 -0.50

-6.3 225.0 -3.1+

-2.7 225.0 -0.8

TABLE 4.3. Percentage of individual secondary structure present

in peptides 1 to lj. calculated by the method described

in methods and materials.

Peptide S01vent Sec0ndary Structures

a -Helix f3 -Beta thordered

TFE 0.35 0.17 0.48

TFE/H2

0 (2: I) 0.28 0.21 0.51 1 - SDS 0.14 0.41 0.45

Brij 35 0.70 0.30

lFE 0.17 0.26 0.57

lFE/H2

0 (2:1) 0.21 0.22 0.57

2 SOS 0.11 0.33 0.56 -Brij 35 0.66 0.34

Na-OOC 0.69 0.31

lFE 0.55 0.45

lFE/H2

0 (2: 1) 0.52 0.48

MeOH 0.01 0.50 0.49

3 MeOH/H2

0 (3:1) 0.0 I 0.44 0.55

MeOH/H2

0 (l:1) 0.05 0.33 0.62

SOS 0.08 0.40 0.52

SOS/0.5 M NaCI 0.57 0.43

lFE 0.33 0.25 0.42

IF E/H2

0 (2: I) 0.23 0.30 0.47 4

MeOH 0.07 0.45 0.48

MeOH/H 2O (2: 1) 0.48 0.52

SDS 0.23 0.35 0.42

69

6 has negative bands at 209 nm and 220 nm. Secondary structural parameters

determined by the method of Chen (Chen ~ 5!l., 1974) and comparison of the

spectra with reference to spectra of poly-Lys indicates the presence of both

helical and B -structure and 60% unordered conformation.

Peptides !J ~ and 2. The CD spectra of peptide Z corresponding to the amino-terminal region,

peptide ~ corresponding to the central hydrophobic region and peptide 9 corres-

ponding to the carboxy terminal region, of E. coli lipoprotein signal sequence,

in TFE are shown in Fig. 4.14. All the three peptides have a shorter wavelength

negative band at 203 nm. Peptide Z has a longer wavelength negative band at

220 nm, and peptides ~ and 2. at 225 nm. This indicates that even the small

fragments of signal sequences adopt an ordered conformation. Peptide 8 corres-

ponding to the central hydrophobic core has more ordered conformation than

peptides Z and 2., as is evident from the larger intensities of its bands.

4.4 Discussion

Conformational studies on peptide 1-2 indicate the presence of both

a -helix and B -structure with the B -structure dominating in more hydrophobic

environment. Clearly, signal sequences 1.-2 exhibit considerable conformational

flexibility. While there are limitations in the quantitation of secondary structure

from the CD spectra of relatively small peptides based on the methods of Chen

(Chen et 5!l., (974) and Greenfield and Fasman (Greenfield and Fasman, 1969)

and also secondary structure prediction according to Chou and Fasman (Chou

and Fasman, 19n), it is evident that the signal sequences 1.-2 have a significant

amount of B -structure compared to the signal sequences of M 13 coat protein

(shinn"lr and Kaiser, 1984), parathyroid hormone (Katakai and Iizuka, (984),

A -receptor (Briggs and Gierasch, 1984) ~nd chicken lysozyme (Reddy and Nagaraj,

(985). Hence it is unlikely that an a -helical conformation is a common structural

feature that is recognized by components of the cells' export machinery.

70

Genetic studies in~. coli have revealed that a charge in the hydrophobic

core of signal sequences leads to the accumulation of precursor proteins in

the cytoplasm (Emr et al., 1980, Michaelis et ~., "1983). The CD studies on

peptides !!.., corresponding to ~. coli lipoprotein signal sequence with a positive

charge (Lys) in the hydrophobic core shows both a -helix and f3 -structure in

TFE and SDS, similar to peptides 1 and l. Thus, it is unlikely that a mutant

signal sequence with a charge in the hydrophobic core is non functional because

of a conformational change as compared to the wild type signal sequence.

On the other hand peptide !!.. unlike 1-.2. does not adopt f3 -structure in more

hydrophobic micelles like Brij 35 and NaDOC. The inability of peptide 4 to

adopt an ordered conformation is presumably because of the positive charge

in the hydrophobic core which prevents its interaction with more hydrophobic

micelles.

It has been shown that the shorter peptide fragments corresponding

to the A -receptor protein signal sequence, adopt ordered conformation (Reddy

and Nagaraj, 1985). The synthetic peptide fragments of the ~. coli lipoprotein

signal sequence, also adopt ordered conformations, and the fragment correspond-

ing to central hydrophobic core are more ordered with' secondary structural

features also clearly discernable.

The signal sequence of the A -receptor protein in phospholipid mono-

layers has been shown to adopt exclusively f3-structure (Briggs ~ ~., 1985).

E. coli lipoprotein and alkaline phosphatase signal sequences also show 70%

B -structure, with almost no helical content in lTIembrane mimetic environments

like micelles of SDS, Brij 35, Na-cholate and Na-DOC. The tendency of signal

71

sequences to adopt a S-structure in membrane mimetic media, although thermo-

dynamically less favourable compared to that of a -helix (Engelman and Steitz,

1,981), indicates that the transition of signal sequences to a B -structure after

insertion into the membrane may be an important feature of signal sequences,

at least in the initial step of translocation.