The design and construction of synthetic protein mimics

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Pure &Appl. Chern., Vol. 64, No. 8, pp. 1061-1066, 1992.

Printed In Great Britain.

@ 1992 IUPAC

The design and construction of synthetic protein mimics

P. Balaram

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 5 6 0 0 1 2 , India

Abstract

-

A strate y for the modular construction of synthetic protein mimics base% on the ability of non-protein amino acids to act as stereochemical directors of polypeptide chain folding, is described. The use of d - a m i n o i s o b u t y r i c acid (Aib) to construct stereochemically rigid helices is exemplified by crystallographic and spectroscopic studies of several apolar peptides, ranging i n l e n th from seven t o sixteen residues. T h e problem of l i n k e r d e s f g n i n e l a b o r a t i n g

a,&

m o t i f s i s c o n s i d e r e d . Analysis of protein crystal structure data provides a guide to choosing linking sequences. Attempts at constructing linked helical motifs linking Gly-Pro segments are described.

Ths use of f lexii?in!inkers, like € -aminocaproic acid i s e x a m - ined and the crystallographic and solution state analysis of a linked helix motif is presented. T h e use of bulky sidechain modifications o n a helical scaffold, as a means of generatin putative binding sites is exemplified by a crystal structure of a peptide packed in a parallel zipper arrangement.

INTRODUCTION

The synthetic construction of peptides that mimic the supersecondary structural motifs i n proteins requires that suitably designed sequences fold in predictable fashion (ref. 1 ) . Polypeptide sequences employin the twenty genetically coded amino acids possess a degree of structuraf flexibility that makes definitive redictions diffpcult, especially for sequences that have been designed $e novo (ref. 2 , 3 ) . A n approach that is being developed i n this l a b o r a t m y m o y s non-protein amino acids as stereochemical directors of olypeptide chain folding (ref. 4 , 5 ) . I n this strategy conformationalfy rigid modules of secondary structure

,

like helices, are constructed from sequences containing amino acids w i t h overwhelmingly strong stereochemical preferences. For instance, d - a m i n o i s o b u t y r i c a c i d (Aib) a c o m m o n c o n s t i t u e n t of m a n y f u n g a l peptide antibiotics (ref. 6 ) facilitates helix formation i n oligopep- tides and stabilises 3 U -Gelical structures i n relatively short peptides (ref. 6,7). The availability of helical modules then permits further elaboration of se uences containing l i n k e d , secondary structure elements. This strategy

4 0 s

construction of synthetic protein mimics i s schematically illustrated i n Fig. 1. The k e y steps i n this approach are :

Choice of ap ro riate structural motif.

Design, syntgeses and characterisation of structured modules.

Syntgesis and structural characterisation of protein mimics, iii) Desi n of linking segment sequences.

iv)

+ I

Fig. 1.

A modular ‘Meccano set‘

approach to synthetic protein design.

Schematic illustration of O ( , N -motif construction.

aQ!

1061

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even though the central Pro residue interrupts a regular chain of i n t r a - molecular hydrogen bonds (ref. 11). Similarly, a n overall helical fold

A i b n = 1 Deg n = 4 Ac5c

n = Z D p g n = 5 Ac6c

Fig. 2. Non-prot.ein amino acid residues. Aib,O(-aminoisobutyric a c i d ; D e g , diethylglycine; dipropylglycine. A c ~ c , 1-aminoc clo- pentane carboxylic ac?$!’Ac6c, 1-aminocyciohexane carboxyiic acid.

is maintained in the C-terminal segment of the fungal peptide zervamicin (ref. 12) and a s nthetic analog (ref. 1,3), despite the presence of a s many as three ProYHyp residues. Aib residues c a n therefore be used to romote helical structures, a property that has been used to advanta e fn extensive studies of peptide helices i n crystals and solution. T g e structural characterisation, at hi h resolution

<

^-1.0

1,

of helical peptides in single crystals has afforded many n e w insights into helix packing, hydration and conformational heterogeneity i n crystals (ref.

7). Helices ranging in length from 7 to 16 residues have been characte- rised in apolar sequences containing Aib residues. Structures which incorporate 3 to 4 helical turns i n their crystal state conformations include the peptides Boc-(V-A-L-U) -0Me (ref. 14), Boc-V-A-L-U-V-A-k-V-A -L-U-V-A-L-U-OMe (ref. 14) and Boc$U-(V-A-L-U) -0Me (ref. 15). (Single letter amino acid code: V = V a l , A= A l a , L = L e a , U=.Aib). CD spectra characteristic ol: highly helical sequences are obtained i n methanol s o -

lutions, su gesting that solid state and solution conEormations are similar (ref. 16).

a m i n o a c i d s c o n t a i n i n g l o n g e r a l k y l s i d e c h a i n s l i k e d i e t h y l l y c i n e (Deg), d i p r o p y l g l y c i n e ( D p g ) a n d d i b u t y l g l y c i n e (Dbg). S t u j i e s of homooligomers i n crystals and theoretical calculations have suggested that D e g , Dpg and Dbg favour Eully extended (C ) conformations.

Preliminary investigations o f tripeptides of the tyae Roc-Ala-X-Ala-OMe have shown that for X = D p g , Dbg extended forms are f a v o u r e d , whereas for X = Acnc ( n = 6,7), P - t u r n or folded structures are referred (ref.

17-20). While characterisation of longer se uences contafning DPg and Dbg are awaited, it is clear that these resi8ues may be

cing polypeptide chains to extended €orms.

Far fewer studies have been carried out o n the Cdu-dialkylated

useful i n f o r -

L I N K I N G HELICES

While stereochemically rigid modules like helices and disulfide bridged antiparallel hairpins (ref. 21) may be constructed, further a s s e m b l y r e q u i r e s t h e d e s i n of l i n k i n g pe t i d e s e q u e n c e s t h a t a r e necessary to connect indivifual structural efements. Analysis of the amino acid compositions and backbone conformations of short linking loops in crystalline proteins, provides a means of designing linkers.

Using a 6 5 protein data s e t , all d d , p f i , c Y B and @ # motifs containing 1 to 5 residue linking segments have been examined. Fig. 3 illustrates results of such an anal sis for do( motifs (ref. 22). T h e identifica- tion of linkers that fall into well defined conformational families

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Synthetic protein mimics 1063

271

(b) (d 1 ( f ) ( h )

F i g . 3 . Conformational families observed for linking loops indp(motifs,

represented on a (

# , v )

^plot. CaC tracings of representative motifs with residue numbers indicated are shown below each plot.

The numbers 1 -5 i n the 0 , V ) diagrams mark the conformations Residues in the l i n - king loops are marked with a n asterix i n the C d tracings.

(a) single residue linkers, (b) 2 4 2 - 2 7 1 cytochrome c peroxidase at residues 1 - 5 of the

i

inking segment.

,

(c) two residue linkers, (d) R 7 9 - K l 0 4 trp-repressor three residue l i n k e r s , (f) 7 6 - 1 1 1 erythrocyyorin five residue linkers, (h) 8 9 - 1 1 7 citrate synthase rom ref. 2 2 ) .

0 1 3

Fig. 4 . (left) Crystal state conformation of B o c - V a l - A l a - L e u - A i b - V a l - A l a - L e u - A c . p - V a l - A l a - L e u - A i b - V a l - A l a - L e u - O M e , illustrating d i s - placed helical modules (from ref. 2 5 ) , (ri ht) Crystal s t r u c - ture of Boc-Aib-Glu( O B z l ) -Leu-Aib-Ala-Leu-fib-Ala-Lvs

-

( Z ) ^. ^.-Aib- OMe illFstrating ins$rtion of sidechains f r o m ad'acent columns into a binding site

(from ref. 2 7 ) . cavity on a neighbour (maried i n black)

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pattern as judged by 2D-NMR, CD and molecular dimensions obtained from a determination of cell parameters of single crystals i n the space group P 4 (ref. 1 6 24). I n d e e d a l l d a t a w e r e c o n s i s t e n t w i t h a l a r g e l y coJtinuous helix, despite the presence of 8 contiguous non-Aib residues, including the potentially helix breaking Gly-Pro unit. Ironically, this result sug ests that breaking a long helical segment nucleated by even a couple of l i b residues may not be an easy proposition. A straight f o r - ward approach to this problem would be to enhance the flexibility of the l i n k e r . T h i s w a s a c h i e v e d by u s i n g E - a m i n o c a p r o i c a c i d (Acp) a s a linker i n the peptide B o c - V a l - A l a - L e u - A i b - V a l - A l a - L e u - A c p - V a l - A l a - L e u - Aib-Val-Ala-Leu-OMe (1). The conformation i n crystals i s s h o w n i n F i 4 (ref. 2 5 ) . It i s clearly seen that both heptapeptide segments retag:

their helical conformations, However, the axes of the two cylindrical helical modules are displaced by the Acp residue although the two i n d i - vidual helices remain approximately parallel, but extended. The central CO (Ala(6), Leu(7)) and NH (Va1(9), Ala(l0)) groups wh+ch do not form intramolecular hydrogen bonds interact intermolecularly with corre- s ondin donor and acceptor groups on adjacent molecules, stabilising tge con5ormation observed in crystals, Interestingly, the HPLC retention time of peptide 1 o n a reverse hase C column i s appreciabl lower than that of single continuous hefices of'%omparable size. Fig.

5

HPLC

-

^C18^{( 5 p )}

80-95'l. MeOH-H$ 15' 16 Detection 226 nrn

12 20 28 3 6

' 4

TIME ( m i n )

Fig. 5 . HPLC trace of a peptide mixture of varying l e n g t h , indicated above the schematic cylindrical structures. Retention times marked above the peaks.

-Val-Ala-Leu-Aib-Val-Ala-Leu-. Interconversions between antiparallel, close acked cylinders and o p e n , extended forms i s illustrated at tge bottom.

Peptide abbreviation used i s UV7 =

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Synthetic protein mimics 1065

shows a chromatogram of a peptide mixture of helical eptides, of varying length and similar sequence, whose rodlike helfcal conformations have been established i n both the solid state and solution. Peptide 1 has a d r a m a t i c a l l y l o w e r r e t e n t i o n t i m e a s c o m p a r e d t o c y l i n d r i c a l molecules with nearly the same number of residues. This suggests that t,he e x p o s e d s u r f a c e a r e a , a c c e s s i b l e f o r i n t e r a c t i o n s w i t h thee,#

chains on the column, is appreciably lower. This observation favours a compact conformation i n which the two helical modules close ack i n antiparallel fashion as shown schematically i n Fig. 5. T h e pacting of large complementary surfaces i n organic solvents c a n indeed be driven by solvophobic interactions (ref. 26). Modelling studies indicate that rotation about the single bonds of the flexible pentamethylene chain of the Acp linker suffices to reorient the helices comfortably. Indeed, the present results suggest a degree of structural flexibility i n 1 with the orientation of the helical modules being determined by environmental factors. A 31-residue pe tide containin four helical modules and three linking Acp residues B o c - ~ V a l - A l a - L e u - A i % - V a l - A l a - L e u - A c p ) 3 - V a l - A l a - L e u - Aib-Val-Ala-Leu-OMe has been synthesised and conformational characterisation is i n progress.

electrostatic intereactions between the helical modules. A s a first attempt i n t,his direction, the 18-residue pe tide Boc-Aib-Ala-AibyLeu- Ai b -L y s -V a 1 -Leu -G 1 y -P r o -As p -A1 a -Leu

-

^Ai^b^-A1^a

-

^{Ai% -Leu}

-

^Ai^b

-

OMe was d e s ¹gne d with a central Gly-Pro linking unit. A comparison of CD spectra of both the charged peptide and the protected analog (Lys (Z) and Glu (OBzl)) revealed very similar helical conformations. I n d e e d , the CD data w e r e consistent with a continuous helical conformation of both molecules.

During the course of studies aimed at building up helical modules w i t h a potential for charge introduction, the sidechain protected peptide Boc- A i b - G l u ( O B z l ) - L e u - A i b - A l a - L e u - A i b - A l a - L y s ( Z ~ ) - A i b - O M e was s nthesised and cr s t a l l i s e d . T h e c r y s t a l s t r u c t u r e ( F i g . 4 ) r e v e a l e 8 a p e r f e c t l y helical conformation for the molecule w i t h a novel packing arrangement (ref. 27). T h e bulky protected sidechains of Lys and G l u project per- pendicular to the helix axis resulting i n the formation of a lar e cup- P o r t i ~ n s of adjacent helical columns intru%e into this ‘putative binding site

.

T h e arrangement of h e l i c e s , head-to-tail within columns which are parallely packed, gives rise to a novel zipper like association o f adjacent columns. This structure sug ests that the residue separation o f the bulky sidechains i s optimal for creating a potential ‘host‘ site. I n d e e d , a heptad repeat of the smaller isobut 1 zidechains of LTU residues o n a helical backbone is a feature of tge knobs in holes packing (re€. 2 8 ) envisa ed for leucine z i p ers (ref.

29) and coiled coil proteins (ref. 30). %he structure descrfbed above suggests that construction of orthogonal pe tides by means of functiona- lised sidechains could lead to n e w molecopes w i t h interesting binding properties. Ongoing studies i n this laboratory are aimed at exploring such structures. The resent approach aims at develo ing a well o r g a - nised molecular scaf f o f d , using defined polypeptide c i a i n folding a t - terns, as a means of controlling spatial orientation of reactive s f d e - chains. The use of hydrophobic helical modules results i n relative1 hi h solubility in or anic solvents, of peptides of considerable lengtg Thfs facilitates studfes of foldin under conditions where the dominan;

interactions can be modelled and fydrophobic effects are absent. T h e construction o € amphi.pathic sequences should permit extension to aqueous solutions w i t h inter-module association being driven by hydrophobic interactions.

The control of helix orientation may be achieved by building i n

i k e , acyclic cavity.

Acknowledgements I am deeply indebted to Dr. Isabella Karle (Naval

Research Laboratory, Washington D.C. U.S.A.) for her continued collaboration i n this project, and Dr. K. U m a and Dr. M. Sukumar f o r their extensive studies over several years. T h e protein data analysis has been carried out i n collaboration with R. Sowdhamini, Dr. N. Srinivasan and Dr. C. Ramakrishnan. Financial support from the Department of S c i - ence and Technology, Government of I n d i a , is gratefully acknowledged.

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