Photolytic control of peptide self-assembly

Article abstract of DOI:10.1021/ja035360b

Herein, we present a methodology that allows for the temporal control of fibrillization of amyloidogenic peptides. This general approach implements a photolabile linker that connects the amyloidogenic peptide to a fibril-inhibitory unit, in this case, a p

Full text of DOI:10.1021/ja035360b

Published on Web 06/03/2003
Photolytic Control of Peptide Self-Assembly
Carlos J. Bosques and Barbara Imperiali*
Departments of Chemistry and Biology, Massachusetts Institute of Technology,
77 Massachusetts AVenue, Cambridge, Massachusetts 02139
Received March 28, 2003; E-mail: imper@mit.edu
Scheme 1. Modular Design of the System for Studying Photolytic
The self-assembly of peptides and proteins into fibrils is one of
Control of Peptide Self-Assemblya
the major events leading to the development of many neurodegen-
erative diseases.1-3 This natural process has served as an inspiration
to the field of biomaterials as the independent organization of
biomolecules into supramolecular structures provides a powerful
approach for the generation of new materials with potentially
interesting properties.4-7 Unfortunately, in most systems, the
spontaneous self-assembly process can be very difficult to control.
In addition, the uncontrolled polymerization can represent a
significant obstacle to the preparation and purification of the system
for study. Hence, temporal control of peptide self-assembly is
desired, and, ideally, it would be advantageous if a peptide could
be synthesized and chemically modified in a soluble and stable
form until the fibrillization is triggered, without the need for
additional manipulations. Herein, we report a method for the
temporal control of peptide self-assembly into fibrils using an
amyloidogenic peptide derived from the human prion protein (PrP)
fragment 174-195.8,9 The design (Scheme 1) implements a synthetic
photolabile linker and a “fibril-inhibitory unit” that stabilizes the
peptide as a soluble species for at least 12 h. Upon photolysis, the
fibril-inhibitory unit and photolinker are detached from the amy-
loidogenic peptide, which is then capable of self-assembly. Previ-
ously, other methods including divalent cation release utilizing
stimuli-responsive liposomes have been implemented to trigger
peptide self-assembly.10
Photolabile linkers have been used in many applications including
solid-phase peptide synthesis, for the facile cleavage of peptides
from the solid support,11 as well as for the controlled release of
metal ions12 and bioactive molecules such as peptides and nucleic
acids.13 Caged peptides provide an efficient approach for the
controlled exposure of active epitopes upon activation with UV
irradiation. Unfortunately, the commercially available photolinkers
for peptide applications are only compatible with introduction at
the C-terminus of a peptide.11 If placed at the N-terminus or in the
side chain of an amino acid, the photodissociation step releases a
non-native species with part of the photoproduct still attached to
the N-terminus or the side chain of the peptide, which may not be
desirable for further studies. An example of such a situation is in
studies on the PrP 174-195 fragment, wherein fibrillization of the
peptide is very sensitive to the electrostatic environment near the
N-terminus. Therefore, to transiently stabilize the amyloidogenic
peptide in a soluble form, the photolinker and a fibril-inhibitory
unit should be placed near the N-terminus. To achieve this, the
commercially available photolabile linker 3-amino-3-(2-nitrophen-
yl)-propionic acid (ANP) was reconfigured to allow the attachment
via an amide linkage to the aspartic acid side chain of the N-terminal
residue of the amyloidogenic peptide (Chart 1). This mode of
attachment allows the photolytic release of the intact native peptide
2 (without photolysis side products attached) and leaves unmodified
asparagine (Asn 174) at the N-terminus (Chart 1). N,N-Dimethyl-
ethylenediamine (DMDA) groups were employed as the fibril-
a Scale bar ) 200 nm.
Chart 1. Structures of Peptides 1 and 2
inhibitory units. DMDA groups have been previously used to sta-
bilize amyloidogenic peptides in solution due to the positive charge
of these groups at neutral pH (DMDA pKa 10-11).5 Additionally,
the low reactivity of tertiary amines is important in photolysis.14
Thus, the DMDA unit is an excellent candidate for the solubilization
of the amyloidogenic peptide. Therefore, an asparagine analogue
incorporating the DMDA functionality in the side chain was
synthesized for facile incorporation of the DMDA groups into the
peptide during SPPS. The photolinker and the Asn-DMDA mono-
mers were easily attached to the side chain of the N-terminal residue
to generate peptide 1.
To determine if irradiation of peptide 1 would afford the desired
photoproducts, and also to establish the time frame of the reaction,
the peptide was dissolved in water (70 µM peptide, 1 mM DTT)
and exposed to UV-vis irradiation from a transilluminator (365
nm, 7.3 mW/cm2).15 The reaction was monitored at different
irradiation times by analyzing aliquots from the solution by reverse
phase HPLC (Figure 1A). The generation of the expected photo-
product (peptide 2) was confirmed by ESMS. After 15 min of
irradiation, complete disappearance of the starting material was
observed with no further increase in peptide 2.
To test the design and the concept that the fibril-inhibitory unit
could provide the desired stabilization of the peptide until photolytic
cleavage, fibrillization kinetics for the photolyzed and unphotolyzed
peptide 1 and the independently synthesized peptide 2 were
compared. The peptides (120 µM) were dissolved in water with 1
mM DTT. Peptide 1 was either kept in the dark or exposed to UV
light from a transilluminator for 15 min. All samples were then
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J. AM. CHEM. SOC. 2003, 125, 7530-7531
10.1021/ja035360b CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
that fibrillization of peptide 1 is significantly delayed and only a
small density of fibrils is observed after 24 h of incubation. In
contrast, for the photolyzed peptide sample, fibrils are observed in
the EM analysis performed at 6 h. The control peptide 2 showed
small filaments at time zero, and extended fibrils were observed
by 6 h. These results are consistent with the aggregation kinetics
and show the photolysis-dependent fibrillization of the amy-
loidogenic construct 1.
Herein, we have presented a methodology that allows for the
temporal control of fibrillization of amyloidogenic peptides. This
general approach should be of value in a variety of studies where
spatial and temporal control of supramolecular association processes
is desired. In this study, we have used the electrostatic properties
of the DMDA group as the key parameter to stabilize the soluble
form of the amyloidogenic peptide until dissociated by photolysis.
The linker unit and the general methodology allow the facile
incorporation of any “inhibitory unit” desired, depending on the
properties of the specific amyloidogenic peptide of interest. For
example, sterically encumbering organic moieties or hydrophilic
species such as carbohydrates can be used for the modification to
inhibit the fibril formation.17 When fibrillization is desired, the
peptide can be exposed to UV radiation and submitted to conditions
that promote self-assembly.
Figure 1. (A) HPLC traces for the photolysis of peptide 1 to produce
peptide 2. (B) Aggregation kinetics for 60 µM photolyzed (black) and
unphotolyzed (red) peptide 1 and peptide 2 (blue) at pH 7.5.
Acknowledgment. This work was supported by the NIH
(GM-39334).
Supporting Information Available: Synthesis and characterization
of the photolabile linker, asparagine-DMDA, and peptide. Aggregation
kinetics of peptide 1 at different pH and control experiments (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. Time-dependent EM analysis for 60 µM peptides incubated at
pH 7.5 with schematic representation of the species present in the solutions.
Peptide 1 (top); photolyzed peptide 1 (middle); peptide 2 (bottom). Bar
size ) 200 nm.
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