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 len th from seven t o sixteen residues.
The 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 is examined and the crystallographic and solution state analysis of a
linked helix motif is presented.
The 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 ) .
An 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 ) .
In
this strategy conformationalfy rigid modules of secondary
structure ,
like helices, are constructed from sequences containing amino acids with
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 oligopeptides 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 linked, secondary structure
elements. This strategy
construction of synthetic protein mimics is
schematically illustrated i n Fig. 1.
The key steps i n this approach
are :
40s
Choice of ap ro riate structural motif.
Design, syntgeses and characterisation of structured modules.
iii) Desi n of linking segment sequences.
iv) Syntgesis and structural characterisation of protein mimics,
+
Fig. 1.
A modular ‘Meccano set‘
approach to synthetic
protein design.
Schematic illustration
of O ( , N -motif construction.
I
aQ!
1061
P. BALARAM
1062
N O N - P R O T E I N A M I N O ACIDS IN PEPTIDE D E S I G N
Fi ure 2 lists the structures of some non-protein amino acid residues taat a r e b e i n g i n v e s t i g a t e d .
Aib (ref. 6,7) and the 1 a m i n o c y c l o a l k a n e - 1 - c a r b o x y l i c acid (Acoc) (ref. 8 9 ) residues
EavoBr heical cozf orma&ions , with backbone dihedral angles of
+ 3 0 , ?y - 2 50 & 3 0
Indeed i n the case of Aib, even incorporation
of a sin le residue int.0 a heptape tide (ref. lo) or hexapeptide (ref.
11) resufts in the stabilisation o f helical conformations i n crystals.
A particularly dramatic example is the peptide B o c - V a l - V a l - A i b - P r o - V a l Val-Val-OMe, where a 3 1 0 -helix is observed i n the crystalline state,
even though the central Pro residue interrupts a regular chain of intramolecular hydrogen bonds (ref. 11).
Similarly, a n overall helical fold
Aib
Fig. 2.
$sfi~nf$
.
-
n = 1 Deg
n=ZDpg
n = 4 Ac5c
n = 5 Ac6c
Non-prot.ein amino acid residues. Aib,O(-aminoisobutyric acid;
D e g , diethylglycine;
dipropylglycine. A c ~ c ,1-aminoc clopentane carboxylic ac?$!’Ac6c,
1-aminocyciohexane carboxyiic
acid.
is maintained in the C-terminal segment of the fungal peptide zervamicin
(ref. 12) and a s nthetic analog (ref. 1,3), despite the presence of as
many as three ProYHyp residues.
Aib residues can therefore be used to
romote helical structures, a property that has been used to advanta e
fn extensive studies of peptide helices i n crystals and solution.
Tge
structural characterisation, at hi h resolution < -1.0 1, of helical
peptides in single crystals has afforded many new insights into helix
packing, hydration and conformational heterogeneity i n crystals (ref.
7).
Helices ranging in length from 7 to 16 residues have been characterised in apolar sequences containing Aib residues.
Structures which
incorporate 3 to 4 helical turns i n their crystal state conformations
include the peptides Boc-(V-A-L-U) -0Me (ref. 14), Boc-V-A-L-U-V-A-k-V-A
-L-U-V-A-L-U-OMe (ref. 14) and Boc$U-(V-A-L-U) -0Me (ref.
15).
(Single
letter amino acid code: V = Val, A= A l a , L= Lea, 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).
Far fewer studies have been carried out o n the
Cdu-dialkylated
amino acids containing longer alkyl sidechains like diethyl lycine
(Deg), d i p r o p y l g l y c i n e (Dpg) and 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 favoured, 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 useful i n forcing polypeptide chains to extended €orms.
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 the 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 dd, 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).
The identification of linkers that fall into well defined conformational families
Synthetic protein mimics
1063
271
Fig.3.
(b)
(d 1
(f)
(h)
Conformational families observed for linking loops indp(motifs,
represented on a (
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
at residues 1 - 5 of the inking segment. Residues in the linking 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
, (c) two residue linkers, (d) R 7 9 - K l 0 4 trp-repressor
three residue linkers, (f) 7 6 - 1 1 1 erythrocyyorin
five residue linkers, (h) 8 9 - 1 1 7 citrate synthase
rom ref. 2 2 ) .
#,v)
i
013
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 Ala-Leu-Ac.p-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe,
illustrating displaced helical modules (from ref. 2 5 ) , (ri ht) Crystal struc-
ture of Boc-Aib-Glu( O B z l ) -Leu-Aib-Ala-Leu-fib-Ala-Lvs
- (.Z ). -AibOMe illFstrating ins$rtion of sidechains from ad'acent columns
into a binding site cavity on a neighbour (maried i n black)
(from ref. 2 7 ) .
1064
P. BALARAM
sug ests that one approach to the problem of interhelix orientation
woufd be to use stereochemically constrained amino acids with a strong
This
preference for the. appropriate region of @ , y/ space (ref. 5).
would be particular1 important in desi n of motifs where interhelix
contacts a r e m i n i m a l a s i n the c a s e o f n e a r l y o r t h o g o n a 1 , d H m o t i f s
The results also show a clear preference of some amino
(ref. 2 2 , 2 3 ) .
acids like Gly for the linking segments.
Initial attempts in this
laboratory to use a central Gly-Pro se ment flanked by two well characterised helices resulted in a 18-resi%ue ieptide Boc-Aib-Val-Ala-LeuAib-Val-Ala-Leu-Gly-Pro-Val-Ala-Leu-Aib-Val- la-Leu-Aib-OMe. This peptide f a i l e d to s h o w a n a p p r e c i a b l e break i n t h e h e l i c a l f o l d i n g
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
24).
Indeed all data were consistent with a largely
P 4 (ref. 1 6
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 forward approach to this problem would be to enhance the flexibility of the
linker.
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 acid (Acp) a s a
linker in 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 in crystals is shown i n Fi
4 (ref. 2 5 ) .
It is 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 individual 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 corres ondin donor and acceptor groups on adjacent molecules, stabilising
tge con5ormation observed in crystals,
Interestingly, the HPLC retenhase C
column is appreciabl
tion time of peptide 1 o n a reverse
lower than that of single continuous hefices of'%omparable
size. Fig.
5
-
HPLC C18 (5p)
80-95'l. MeOH-H$
16
15'
Detection 226 nrn
'
Fig. 5 .
4
12
20
TIME ( m i n )
28
36
HPLC trace of a peptide mixture of varying length, indicated
above the schematic cylindrical structures.
Retention times
marked above the peaks. Peptide abbreviation used i s UV7 =
-Val-Ala-Leu-Aib-Val-Ala-Leu-. Interconversions between antiparallel, close acked cylinders and open, extended forms is
illustrated at tge bottom.
1065
Synthetic protein mimics
shows a chromatogram of a peptide mixture of helical eptides, of varying length and similar sequence, whose rodlike helfcal conformations
have been established in 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 to 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 in which the two helical modules close
ack in
The pacting of
antiparallel fashion as shown schematically in Fig. 5.
large complementary surfaces in organic solvents can 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 in 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 in progress.
The control of helix orientation may be achieved by building in
electrostatic intereactions between the helical modules.
As a first
attempt in t,his direction, the 18-residue pe tide Boc-Aib-Ala-AibyLeuAi b -L y s -V a 1 -Leu -G 1y -P ro -As p -A1 a -Leu Ai b -A1 a Ai% -Leu Ai b OMe was d e s 1 gne 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.
Indeed, the CD data were
consistent with a continuous helical conformation of both molecules.
During the course of studies aimed at building up helical modules with a
potential for charge introduction, the sidechain protected peptide BocAib-Glu(OBzl)-Leu-Aib-Ala-Leu-Aib-Ala-Lys(Z~)-Aib-OMe
was s nthesised and
cr stallised.
T h e c r y s t a l s t r u c t u r e (Fig. 4 ) r e v e a l e 8 a p e r f e c t l y
helical conformation for the molecule with a novel packing arrangement
(ref. 27).
The bulky protected sidechains of Lys and Glu project perpendicular to the helix axis resulting in the formation of a lar e cupike, acyclic cavity. P o r t i ~ n sof adjacent helical columns intru%e into
The arrangement of helices, head-to-tail
this ‘putative binding site
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 is optimal for creating a
potential ‘host‘ site. Indeed, a heptad repeat of the smaller isobut 1
zidechains of LTU residues on a helical backbone is a feature of tge
knobs in holes packing (re€. 2 8 ) envisa ed for leucine zip ers (ref.
29) and coiled coil proteins (ref. 30).
%he structure descrfbed above
suggests that construction of orthogonal pe tides by means of functionalised sidechains could lead to new molecopes with interesting binding
properties.
Ongoing studies in this laboratory are aimed at exploring
such structures.
The resent approach aims at develo ing a well organised molecular scaf fofd, using defined polypeptide ciain folding atterns, as a means of controlling spatial orientation of reactive sfdechains.
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.
The
construction o € amphi.pathic sequences should permit extension to aqueous
solutions with inter-module association being driven by hydrophobic
interactions.
-
-
-
-
.
I am deeply indebted to Dr. Isabella Karle (Naval
Research Laboratory, Washington D.C. U.S.A.)
for her continued collaboration in this project, and Dr. K. Uma and Dr. M. Sukumar for their
extensive studies over several years. The protein data analysis has
been carried out in collaboration with R. Sowdhamini, Dr. N. Srinivasan
and Dr. C. Ramakrishnan. Financial support from the Department of Science and Technology, Government of India, is gratefully acknowledged.
Acknowledgements
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