Protein design enables the

stabilization of a transient
molecular state
By David K. Romney and Scott J. Miller

he description of the potential energy

surface of a single bond rotation is a
standard concept for understanding
chemical reactions and molecular motions (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 coordinate diagram or multidimensional potential 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 energy landscape (4).
It can be very difficult to observe the
high-energy states along a complex trajectory. Ultrafast spectroscopy provides
one lens with which to see fleeting highenergy states (5), but many other techniques 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 approaches to artificial protein design to stabilize a high-energy state of a simple but
dynamic molecule. Their success is attributable to the creative combination of computational design, the use of an amino acid
not found in natural proteins, and the iterative synthesis and crystallographic evaluation of candidate structures.
The fundamental question posed by
Pearson et al. is whether the packing forces
of a protein can distort intrinsic bond rotations in a molecule to such an extent that
an apparent bond-rotational transition
state geometry may be observed. Using biphenyl 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 conclusion. First, biphenyl is not found in extant
proteins. The authors accomplished its
Department of Chemistry, Yale University, New Haven, CT
06520, USA. E-mail: scott.miller@yale.edu

= 45
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

E (kcal/mol)

Climbing
Jacobs ladder

45

(degrees)

90

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.

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 Rosetta to craft the protein environment surrounding the biphenyl (7).
Through several iterative rounds of design and analysis, Pearson et al. collected
data sets that showed increasingly distorted
biphenyl rings, with dihedral angles nearing 0. Computational design, synthesis, and
measurement, informed by visual inspection
of the computational and crystallographic
output, finally led to the targeted protein.
Planar conformation stabilized
by protein packing

Designated BIF_0, it contains a biphenyl

moiety in the recesses of the protein coat,
with its two phenyl rings essentially coplanar (see the second figure).
This observation is remarkable. One can
certainly mine the Protein Data Bank to capture 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 chemical intuition suggests that the observation
of a planar biphenyl requires extraordinary
circumstances. The rational design and synthesis 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 otherwise fleeting molecular events. The observation of features of catalyst-substrate complex
in a carrier protein provides another example (9); other studies will surely follow.
A particularly notable connection made by
Pearson et al. is the possible analogy to transition state stabilization in enzyme catalysis.
The suggestion recalls the Pauling paradigm
for enzymatic rate acceleration, which calls
for the complementarity of an enzymes active 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 features of the biphenyl bond rotational transition 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 energetic compensations required to capture
it. However, biphenyl has a relatively low
barrier to rotation; one challenge in the future will be to apply the approach to the stabilization of even higher-energy structures.
The present accomplishment illustrates
the precision with which proteins may be
designed for functional purposes with computational 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 behold at its top rung, tell of much more to see
in the future.
REFERENCES

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.

SCIENCE sciencemag.org

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
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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.
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10.1126/science.aaa5623
20 FEBRUARY 2015 VOL 347 ISSUE 6224

Published by AAAS

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CHEMISTRY

Climbing Jacob's ladder

David K. Romney and Scott J. Miller
Science 347, 829 (2015);
DOI: 10.1126/science.aaa5623

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