Essay:
Protein Encapsulation within
Synthetic Molecular Hosts
Daishi Fujita
I feel that naturally, in every chemist, there exists a
desire to artificially modify or create new functions or
properties of proteins, being that they are essential
natural compounds. However, this is a daunting
challenge since proteins are very unmanageable under
most conditions in organic chemistry. One significant
issue is stability. Proteins easily lose their function from
heat, suboptimal pH, solvent conditions, etc. Moreover,
since proteins consist of repeats of amino acids,
chemo-selective modification is generally very difficult.
So, there comes an idea to lay out a scaffold around a
protein in the same manner as a construction site around
a building. If we can take advantage of a scaffold, we may
realize customizable protein functionalization.
The scaffolding approach has become a vibrant and active field in organic supramolecular
chemistry. Since the first reports of crown ether and cyclodextrin, a number of nicely
designed molecules that can capture or encapsulate small guest molecules have been
developed and their unique properties demonstrated. However, despite the tremendous
efforts and the rapid growing of host–guest chemistry, the size of target guests is still
limited to that of ions or small molecules. Even today, the biggest guest molecules that can
be enclosed are various fullerenes with diameters of 0.7 nm at most (1) (Fig. 1). Proteins
usually at least have diameters of 4-5 nm, which is incommensurably larger than common
targets. Currently, no organic chemist can come close to scaffolding a compound the size of a
protein. –––– The size difference of the encapsulation of small molecules and proteins is as
different as "gift wrapping" and "house wrapping". A new methodology must be developed
for proteins. This is a challenge which does not lie on the simple extension of conventional
host-guest chemistry.
Fig. 1
Size comparison of common host molecules.
My approach toward this unprecedented challenge in synthesis is the utilization of
self-assembly. Though the synthetic difficulties generally increase exponentially with
molecular weight, we can minimize this effort by making use of self-assembly mechanisms.
One of the largest synthetic molecules reported to date is a palladium coordination nanocage
from our group (Fig. 1). This nanocage is self-assembled from 12 Pd(II) ions (M) and 24 bent
ligands (L). The original size of the M12L24 nanocage is 4.5 nm in diameter. However, I
succeeded in expanding it up to 7.3 nm to deal with the purpose of protein enclosure (2). For
my thesis work, I developed a confirmed bottom-up strategy rather than one which depends
on stochastic encapsulation to realize a highly accurate structure. A protein was attached to
one bidentate ligand covalently and upon addition of Pd(II) ions and additional bidentate
ligands, the M12L24 nanocage self-assembled around the protein (Fig. 2). A sugar pendant for
the ligand is a trick to avoid protein denaturation. It was designed to mildly wrap up the
hydrophilic protein surface (Fig. 2). The combination of above-mentioned strategies and
techniques enabled me to be the first person to manipulate protein-metallorganic
composites as a desecrate molecule.
Fig. 2
Schematic representation of the encapsulation of ubiquitin.
In addition to the significance as an unprecedented challenge in synthesis, this M12L24
scaffolding approach has the potential of being a promising platform for chemistry in next
decades. There have actually been a number of publications that report protein
encapsulation (3). However, their common approach that utilizes organic or inorganic
polymeric media cannot avoid disarrayed and uncontrolled structures in principal. Current
structural analysis methods have been limited to nothing better than electrophoresis or
electron microscopy. In contrast, my M12L24 scaffolding system inimitably provides atomic
level accuracy which realizes the long-awaited next step in structural analysis. With this
accurate scaffolding system in hand, we scientists can now clearly envision the following
applications:
1) Substantial enhancement of protein stability via encapsulation: Minimal
enzyme structure where only the catalytic site is stabilized within the cage.
2) Scaffold modification to manipulate outer environment; Pinpoint
modifications can alter protein functions.
3) Outer frame can facilitate protein crystallization; Stabilizing and therefore
crystallizing proteins which have not been able to do so.
In this regard, the M12L24 scaffolding approach makes a clear distinction from other reported
ill-defined host media.
Fig. 3
DOSY NMR spectra and analytical ultracentrifugation experiments.
In my thesis work, I successfully prepared M12L24 coordination nanocage encapsulate
proteins, as depicted, and perfectly proved their formation with several highly reliable
methods in structural analysis (4). As a protein target, ubiquitin (8.6 kDa, approximately 4
nm in diameter) that has an important role in proteasomal degradation was employed.
Ubiquitin was first tethered to a ligand and with subsequent self-assembly processes it was
enclosed within the M12L24 nanocage. The tertiary structure of the encapsulated ubiquitin
was evaluated by 2D NMR measurements with isotope-labeled samples and was found to
retain its original structure. NMR studies also gave information about the compound in
solution through diffusion coefficient measurement. Diffusion-ordered NMR spectroscopy
(DOSY) confirmed the formation of the giant coordination sphere enclosed ubiquitin (Fig. 3).
Several control experiments were also carefully worked. The analytical ultracentrifugation
(AUC) experiments provided information regarding purity and molecular weight, which
proved that the desired compound was formed in quantitative yield (Fig. 3). Finally, X-ray
crystallography coupled with maximum-entropy method (MEM) analysis visualized both the
nanocage structure and ubiquitin centered in the middle of the cavity. These analytical data
clearly demonstrated that the synthesis of the world’s most accurate caged protein structure
had now been achieved (Fig. 4).
Fig. 4
Single crystal X-ray diffraction analysis.
In conclusion, I have overcome the difficulties of synthesizing protein-metallorganic
composites and achieved a challenging goal during my thesis work. This achievement is not
just one example in a series of other works but the first in a new generation of protein
encapsulation. Supramolecular chemistry, which started with the small ion entrapment by
crown ether about a half century ago, has now developed and expanded to this level. I
believe that my demonstration here marks an important milestone in a wide range of
scientific fields: supramolecular chemistry, coordination chemistry, protein crystallography,
and protein chemistry/biology.
References:
(1) J. R. Nitschke et al. Angew. Chem. Int. Ed. 2011, 50, 3479.
(2) D. Fujita et al. Chem. Lett. 2012, 41, 313.
(3) See the references in (4).
(4) D. Fujita et al. Nature Commun. 2012, 3, 1093.