20 FEBRUARY 2015 VOL 347 ISSUE 6224 829SCIENCE
sciencemag.org
(degrees)
= 45
E (
kc
al/
mo
l)
1.6
1.4
1.2
0.8
0.6
0.4
0.2
0
0 45 90
1
The description of the potential energy
surface of a single bond rotation is a
standard concept for understanding
chemical reactions and molecular mo-
tions ( 1). The energetic progression
around a single bond in biphenyl (see
the first figure) ( 2) provides an illustration.
An entire conformational energy landscape
can be captured with a simple reaction coor-
dinate diagram or multidimensional poten-
tial energy surface ( 3). On page 863 of this
issue, Pearson et al. describe a way to trap
and observe an otherwise fleeting state at a
rarified elevation of the conformational en-
ergy landscape ( 4).
It can be very difficult to observe the
high-energy states along a complex tra-
jectory. Ultrafast spectroscopy provides
one lens with which to see fleeting high-
energy states ( 5), but many other tech-
niques require more stable structures. If a
molecular host can be made to stabilize a
transition state, numerous other analytical
techniques, such as x-ray crystallography,
become available for detailed observation.
Pearson et al. now combine different ap-
proaches to artificial protein design to sta-
bilize a high-energy state of a simple but
dynamic molecule. Their success is attrib-
utable to the creative combination of com-
putational design, the use of an amino acid
not found in natural proteins, and the itera-
tive synthesis and crystallographic evalua-
tion of candidate structures.
The fundamental question posed by
Pearson et al. is whether the packing forces
of a protein can distort intrinsic bond rota-
tions in a molecule to such an extent that
an apparent bond-rotational transition
state geometry may be observed. Using bi-
phenyl as the substructure to address this
question, the authors show the answer to
be a resounding yes. But several hurdles
had to be overcome to reach this conclu-
sion. First, biphenyl is not found in extant
proteins. The authors accomplished its
incorporation into a host protein through
the insertion of the nonproteinogenic
amino acid biphenylalanine using the
amber suppressor tRNA/aminoacyl-tRNA
synthetase pair method ( 6). Second, the
choice of a host protein and, perhaps more
importantly, its required alteration were
not straightforward. The authors used the
computational protein design program Ro-
setta to craft the protein environment sur-
rounding the biphenyl ( 7).
Through several iterative rounds of de-
sign and analysis, Pearson et al. collected
data sets that showed increasingly distorted
biphenyl rings, with dihedral angles near-
ing 0. Computational design, synthesis, and
measurement, informed by visual inspection
of the computational and crystallographic
output, finally led to the targeted protein.
Designated BIF_0, it contains a biphenyl
moiety in the recesses of the protein coat,
with its two phenyl rings essentially copla-
nar (see the second figure).
This observation is remarkable. One can
certainly mine the Protein Data Bank to cap-
ture higher-energy states for rotations about
various types of bonds that depart from
their lowest-energy minima and approach
higher-energy local maxima ( 8), but chemi-
cal intuition suggests that the observation
of a planar biphenyl requires extraordinary
circumstances. The rational design and syn-
thesis of this state in a protein host marks
a singular achievement in molecular design.
The results illustrate the value of protein
hosts for facilitating observation of other-
wise fleeting molecular events. The observa-
tion of features of catalyst-substrate complex
in a carrier protein provides another ex-
ample ( 9); other studies will surely follow.
A particularly notable connection made by
Pearson et al. is the possible analogy to tran-
sition state stabilization in enzyme catalysis.
The suggestion recalls the Pauling paradigm
for enzymatic rate acceleration, which calls
for the complementarity of an enzymes ac-
tive site to the transition state structure of
the catalyzed reaction ( 10).
There is no doubt that Pearson et al. have
observed a substructure that exhibits fea-
tures of the biphenyl bond rotational transi-
tion state. A careful energetic balancing act
is required to stabilize such a high-energy
state in a large molecule. Its persistence is
particularly notable given the sum of the en-
ergetic compensations required to capture
it. However, biphenyl has a relatively low
barrier to rotation; one challenge in the fu-
ture will be to apply the approach to the sta-
bilization of even higher-energy structures.
The present accomplishment illustrates
the precision with which proteins may be
designed for functional purposes with com-
putational methods. Lessons learned here
could well portend applications in protein
and enzyme engineering. The assembly of a
unique ladder to climb, and the sight to be-
hold at its top rung, tell of much more to see
in the future.
REFERENCES
1. J. D. Kemp, K. S. Pitzer, J. Chem. Phys. 4, 749 (1936). 2. A.
Almenningen et al., J. Mol. Struct. 128, 59 (1985). 3. E. V.
Anslyn, D. A. Dougherty, in Modern Physical Organic
Chemistry, E. V. Anslyn, D. A. Dougherty, Eds. (University
Science Books, Sausalito, CA, 2006), pp. 365373.
4. A. D. Pearson et al., Science 347, 863 (2015). 5. J. C.
Polanyi, A. H. Zewail, Acc. Chem. Res. 28, 119 (1995). 6. L. Wang,
A. Brock, B. Herberich, P. G. Schultz, Science 292,
498 (2001). 7. A. Zanghellini et al., Protein Sci. 15, 2785
(2006). 8. A. A. Kossiakoff, S. Shteyn, Nature 311, 582 (1984). 9.
S. Han, B. V. Le, H. S. Hajare, R. H. Baxter, S. J. Miller, J.
Org.
Chem. 79, 8550 (2014). 10. L. Pauling, Nature 161, 707
(1948).
Climbing Jacobs ladder
Transient state. Molecules interconvert between
low-energy conformations by passing through transient
high-energy states. Biphenyl is at a stable energy
minimum when the rings are offset by 45 and a transient
energy maximum when the rings are offset by 0 or 90.
10.1126/science.aaa5623
By David K. Romney and Scott J. Miller
Protein design enables the stabilization of a transient
molecular state
CHEMISTRY
Department of Chemistry, Yale University, New Haven, CT 06520,
USA. E-mail: [email protected]
Planar conformation stabilized by protein packing
Stabilizing an energy maximum. Pearson et al. show
that a carefully designed protein environment enables
observation of a biphenyl moiety in a conformation with
essentially coplanar rings.
Published by AAAS
on M
arch
3, 2
015
ww
w.s
cien
cem
ag.o
rgD
ownl
oade
d fro
m
