ASTROBIOLOGY

Volume 3, Number 3, 2003

© Mary Ann Liebert, Inc.

Methodologies and Techniques for Detecting

Extraterrestrial (Microbial) Life

Terahertz Circular Dichroism Spectroscopy: A Potential

Approach to the In Situ Detection of Life’s Metabolic
and Genetic Machinery

JING XU,1 GERALD J. RAMIAN,1 JHENNY F. GALAN,2 PAVLOS G. SAVVIDIS,1

ANTHONY MICHAEL SCOPATZ,1 ROBERT R. BIRGE,2 S. JAMES ALLEN,1
and KEVIN W. PLAXCO3

ABSTRACT

We propose a terahertz (far-infrared) circular dichroism-based life-detection technology that

may provide a universal and unequivocal spectroscopic signature of living systems regard-
less of their genesis. We argue that, irrespective of the specifics of their chemistry, all life
forms will employ well-structured, chiral, stereochemically pure macromolecules (.500
atoms) as the catalysts with which they perform their metabolic and replicative functions. We
also argue that nearly all such macromolecules will absorb strongly at terahertz frequencies
and exhibit significant circular dichroism, and that this circular dichroism unambiguously
distinguishes biological from abiological materials. Lastly, we describe several approaches to
the fabrication of a terahertz circular dichroism spectrometer and provide preliminary exper-
imental indications of their feasibility. Because terahertz circular dichroism signals arise from
the molecular machinery necessary to carry out life’s metabolic and genetic processes, this
life-detection method differs fundamentally from more well-established approaches based
on the detection of isotopic fractionation, “signature” carbon compounds, disequilibria, or
other by-products of metabolism. Moreover, terahertz circular dichroism spectroscopy detects
this machinery in a manner that makes few, if any, assumptions as to its chemical nature or
the processes that it performs. Key Words: Far-infrared—Submillimeter—Spectroscopy—
Biopolymers—Proteins—DNA—Terahertz—Circular dichroism. Astrobiology 3, 489–504.

INTRODUCTION fostered significant interest in new methods for

the detection of life in extraterrestrial settings

T HE GROW TH OF ASTROBIOLOGY and the rapidly

increasing pace of planetary exploration have
such as Mars, Europa, or Titan (e.g., McKay, 1998;
National Research Council, 2002). The majority of

Departments of 1Physics and 3Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara,
California.
2 Department of Chemistry, The University of Connecticut, Storrs, Connecticut.

489
490 XU ET AL.

these technologies focus on the detection of char- APPROACH

acteristic patterns of organic or inorganic materi-
als (e.g., McKay et al., 1996; Thomas-Keprta et al., It is widely appreciated that all macromole-
2000), the presence of distinctive isotopic frac- cules with ionic or polar constituents absorb
tionations (e.g., Greenwood et al., 1997; Mojzsis strongly across the terahertz part of the spectrum
and Arrhenius, 1998), or the observation of sig- (e.g., Markelz et al., 2000; Globus et al., 2002;
nificant chemical disequilibria (e.g., Sagan et al., Woolard et al., 2002). We have performed simu-
1993). While each of these approaches is a pow- lation studies suggesting that for most, if not all,
erful tool in the quest to detect non-terrestrial life, chiral macromolecules this absorbance will be ac-
they all suffer, at least to some extent, from a ter- companied by relatively strong CD. We have also
restrial bias. For example, on Earth, polycyclic designed and are currently building several CD
aromatic hydrocarbons (PAHs) are invariably as- spectrometers operating at terahertz frequencies.
sociated with highly altered fossils (e.g., fossil fu-
els and their combustion products) and thus are
Simulated TCD spectra
relatively unambiguous signatures of life. Abio-
logical production of PAHs, however, has been While ab initio methods have been used to suc-
suggested for non-terrestrial settings (Zolotov cessfully calculate the CD spectra of small mole-
and Shock, 1999), and PAHs are common in the cules, the direct calculation of CD features aris-
interstellar medium (Henning and Schnaiter, ing from the collective vibrational modes of
1998), thus clouding, for example, the suggestion macromolecules are computationally intractable.
that the PAHs in the Martian meteorite ALH84001 In an effort to develop our intuition about the
are biogenic (McKay et al., 1996). Similarly, non- possible magnitude of the TCD effect we thus re-
biological isotopic fractionation has been ob- sort to calculating the TCD spectra generated by
served (Anbar et al., 2000) that could masquerade two simple physical models of the relevant mol-
as a life signature. Lastly, it is widely held that ecular motions.
inorganic chemical disequilibria led to a false de-
tection event when, by analogy to terrestrial life Mass and spring model. Given the prevalence of
(Klein, 1974), water and simple organic “food” helical substructures in terrestrial biopolymers
were provided to putative Mars organisms in the such as proteins, DNA, and RNA (see Discus-
Viking experiments (Klein, 1992). We may thus sion), we are compelled to explore a “masses and
conclude that most contemporary life-detection springs” model of a helical molecular structure as
approaches are complicated by the need to inter- a simple physical representation that captures the
pret their outcomes in terms of expectations essential features of biological macromolecules.
based on the metabolic processes and products of Here we eschew as much detail as possible and
terrestrial references. strive for the simplest model that produces TCD
We propose here terahertz (far-infrared) circu- and that we can manipulate to develop some in-
lar dichroism (TCD) spectroscopy as perhaps the tuition about spectral signatures and their order
first of a fundamentally different, and thus com- of magnitude.
plementary, class of life-detection methods. This Models of vibrational CD have been explored,
new approach focuses not on the metabolism or but these have largely focused on spectra in
metabolic by-products of life but instead on the the near-infrared, spectra dominated by bond
molecular machinery necessary to carry out life stretching and bending, and the wagging of small
processes. The TCD life-detection approach is subunits in a macromolecule (Deutsch and
based on the argument that stereochemically Moscowitz, 1968, 1970; Holzwarth and Chabay,
pure, chiral macromolecules are a universal and 1972; Schellman, 1973). The terahertz part of the
unambiguous signature of chemical life (Fig. 1), spectrum couples to macromolecular vibrations
and that essentially all such macromolecules will of the “whole” and requires a model that explores
exhibit circular dichroism (CD) over a broad collective vibrations. In collective vibrations near-
range of the terahertz spectrum. We thus believe est neighbor atoms move together, with large rel-
that TCD will detect the molecular machinery of ative motions being experienced only by pairs of
life in a manner that makes few assumptions as atoms that are widely separated in the macro-
to the chemical nature of this machinery or the molecule. An important consideration is that, as
processes that it performs. these low-frequency macromolecular vibrations
TCD FOR SPECTROSCOPIC LIFE DETECTION 491

FIG. 1. The structure of the protein bacteriorhodopsin, a typical example of one of the stereochemically pure,
chiral macromolecules universally associated with life on Earth. Shown is a schematic diagram of the protein in
which only the backbone of the polymer, a few key side chains, and the optical chromophore are illustrated—were
all atoms presented in the figure the protein would appear from the outside to be a well-packed, monolithic struc-
ture. Indicated by arrows are the atomic motions involved in the protein’s low-frequency mode at 0.498 THz. As is
generally the case with the low-frequency vibrational modes of bacteriorhodopsin, this mode involves large ampli-
tude, in-plane motion of its seven a-helices. Our analysis suggests that these low-frequency modes will exhibit sig-
nificant CD. The length of the arrows is proportional to the motion of the element involved, but is exaggerated to fa-
cilitate viewing.

soften, they evolve into large-amplitude confor- parameters used, which roughly approximate
mational changes that are widely held to be bio- the a-helix of terrestrial proteins, are the follow-
logically (functionally) relevant (e.g., Tama and ing: period of the helix, 0.55 nm; masses/period,
Brooks, 2002). 10; charge distribution, 25e/14 sites; highest-
Our model comprises a simple collection of frequency acoustic mode, 10 THz; volume den-
masses distributed uniformly in a helical pattern sity or helix period/m3, 0.9 3 1027 m23 ; relax-
(Fig. 2). Each mass represents more than a sim- ation time, 0.53 3 10212 s.
ple atom or ion; it represents the covalently
bonded atom on the backbone and whatever is Normal mode analysis. While simple, generic
attached to it. We assume that these units inter- models can provide important insights into the
act only with their nearest neighbors. For the sake general issue of macromolecular TCD, it is also
of simplicity we assume all masses are the same, important to explore whether similar effects are
and we treat only the torsional motion of the present in more realistic models of terrestrial
masses about the axis of the helix (Fig. 3). This biopolymers. To this end we have also performed
model will produce acoustic-like torsional detailed calculations of the TCD spectra of a spe-
modes that propagate along the helix. The model cific, representative biopolymer, the archeal pro-
is further simplified by not considering radial tein bacteriorhodopsin (Fig. 1). Normal mode cal-
and longitudinal acoustic modes. To couple to culations (Brooks et al., 1995) were performed to
the propagating terahertz electromagnetic field analyze the low-frequency modes in the native
and produce a polarization that attenuates and protein [wild type (WT)] and a version of the pro-
alters the speed of the electromagnetic wave, tein containing a single mutation in which the
charge must be distributed along the helix. The residue aspartate-96 is replaced with asparagine
492 XU ET AL.

FIG. 2. End and side views of a simple “mass and spring” model that captures the TCD features of a generic,
chiral macromolecule. The motion of the masses is described by a simple rotation about the helix axis. The angular
motion of neighboring masses is coupled through a restoring torque proportional to the relative rotations. An elec-
tromagnetic field forces the charged masses to rotate producing a polarization that can be both in the direction of the
field but also normal to it via the mutual coupling.

(D96N). We selected this mutation because of the and van der Waals interactions switched between
availability of the crystal structure. The atomic co- 1.0 and 1.2 nm.
ordinates used in the calculations were PDB en- Normal modes were obtained using the “Itera-
try 1C3W [0.155 nm resolution (Luecke et al., tive Diagonalization in a Mixed Basis Set” method,
1999b)] for WT and PDB entry 1C8R [0.20 nm res- which was developed previously to handle large
olution (Luecke et al., 1999a)] for D96N. Hydro- molecular systems (Mouawad and Perahia, 1993).
gen atoms were added using Insight II (version This method uses an iterative procedure that pro-
2000, Accelrys, San Diego, CA). The protonation vides good accuracy for the low-frequency re-
states of the residues were assigned following the gions of interest to this study. The program
literature (Ren et al., 2001). The water molecules CHARMM (Brooks et al., 1983; MacKerell et al.,
present in the crystal structure were included in 1998) was used for all energy minimizations and
one set of calculations and excluded in a second normal mode analysis.
set of calculations. This approach allows us to de- The normal modes were determined, and a
termine the impact of biological water on tera- representative low-frequency mode was selected
hertz spectra (Fig. 4). for further analysis (Fig. 1). Analysis of all the
The crystal structure of a protein is generally strong modes below 3 THz indicates that the vast
inappropriate for direct harmonic analysis, and majority involve large-amplitude motion of en-
the structure must be minimized prior to analy- tire helices (similar to those shown in Fig. 1). The
sis (Janezic and Brooks, 1995; Janezic et al., 1995). intensity of such modes is due to the fact that one
We used the program CHARMM (Brooks et al., or more of the residues in these helices are
1983) to generate a minimum energy structure charged, which generates a large change in di-
while applying harmonic constraints to the pro- pole moment associated with the vibrational mo-
tein backbone during the initial minimization. tion. Of particular interest here, however, is the
The constraints were progressively reduced at extent to which such vibrations are CD-active. We
each cycle, and then the constraints were re- simulated the TCD spectrum of a protein under-
moved and the system was minimized until the going large-amplitude motion of helical segments
energy gradient was below 4 3 1026 kJ/mol. The by creating an artificial system containing seven
nonbonded interactions were truncated at 1.3 nm, partially charged masses connected via weak
TCD FOR SPECTROSCOPIC LIFE DETECTION 493

Development of a TCD spectrometer

Sources and detectors. We are currently devel-
oping TCD spectrometers based on two terahertz
sources: the UCSB Free Electron Laser, a tunable,
kilowatt pulsed source covering from 0.12 to 5
THz (Ramian, 1992), and a solid-state, continu-
ous wave (CW), 20 mW Gunn Oscillator operat-
ing at 0.14 THz (Quinstar Technology, Inc., Tor-
rance, CA). The Gunn Oscillator is a compact,
low-mass, low-power source. It is also a CW
source, thus allowing for the rapid modulation of
polarization for the purposes of synchronous de-
tection, which can produce potentially critical im-
provements in signal-to-noise. The free electron
laser, in contrast, fills a three-story building (and
thus is hardly “flight-ready technology”) and is
a non-CW source (repetition rate ,3 Hz). Given
its high output power and tunability, however, it
is an ideal test instrument to explore and docu-
ment TCD in strongly absorbing material over a
broad frequency range. Further, while the tera-
hertz part of the spectrum remains less well de-
veloped than either the UV-visible or near-
infrared parts of the spectrum, much specialized
research instrumentation has been developed,
largely for astrophysical applications. Perhaps
most promising are recent demonstrations of
quantum cascade lasers operating at frequencies
as low as 3.4 THz (Williams et al., 2003) suggest-
ing that compact, robust, low-power solid-state
FIG. 3. Top: Dispersion relation for torsional acoustic
modes of the model helix. The amplitude versus drive fre- terahertz sources will be available within the next
quency, or wavevector, for the particular charge distribu- few years. In addition to high-power sources,
tion in the model helix. Middle: The absorption spectrum strongly absorbing material will also require the
of the model helix. Bottom: The model’s corresponding
use of sensitive detectors and thus cryogenic hot
CD features, defined as the difference between the ex-
tinction of right and left circularly polarized radiation. electron bolometers and photoconductors that
are fast and have NEP (noise equivalent input
powers per root Hz) ,102 12 W. The work de-
bonds. The system was constructed using atoms se- scribed here, however, employs less sensitive, but
lected so that a planar equilibrium geometry is ob- more convenient, room temperature pyroelectric
tained, and the system was minimized using detectors.
Hartree–Fock ab initio procedures and a 6-31G(d)
basis set. The system was then modified by in- Optics for circular polarization. Circular polar-
creasing the masses of the atoms 100–fold to pro- ization occurs when the phase of the transverse
duce resonances that mimic large-amplitude pro- electric field on one axis is advanced or delayed
tein vibrations involving entire helix segments (Fig. by 90° with respect to the (equal amplitude) field
1). The vibrational CD engine within Gaussian-98 on the other axis. To an observer, the net electric
was used to predict the TCD spectrum. The physics field vector will appear to rotate, to the left or
underlying this approach are well established and right depending on which axis is delayed, com-
well documented, and thus we will not reiterate a pleting one rotation per wavelength. Linear po-
detailed discussion of the method here but instead larization can be converted to circular, and vice
direct interested readers to the relevant literature versa, with a birefringent material fabricated as a
(Cheeseman et al., 1996; Frisch et al., 1998). quarter-wave plate or by using wire grid polar-
494 XU ET AL.

izers that separate two orthogonal polarizations

and introduce the appropriate phase delay by a
difference in path length. The “purity” of the
right or left circular polarization depends on the
relative amplitudes of the two phases and is read-
ily assayed (to within a precision of ,1%) by ro-
tating a linear polarizer in the beam and, for some
spectrometer designs, is easily adjusted (via mod-
ulation of the input polarizations). Critically, CD
signal strength is linearly related to polarization
purity, and thus discrepancies of even a few per-
cent are not a significant hurdle with respect to
achieving the qualitative goal of life detection.
The measurement of CD in the visible and
near-infrared (500 nm–10 mm) is well established,
with a number of excellent commercial instru-
ments available (reviewed by Kliger et al., 1990).
In order to generate alternating polarizations
these spectrometers generally employ the elasto-
optic properties to induce birefringence in mate-
rials such as ZnSe. A piezo-electric transducer
provides sufficient drive for a full polarization cy-
cle. A similar approach cannot be employed at FIG. 4. Calculated terahertz spectra of wild-type bac-
terahertz frequencies, however, because the or- teriorhodopsin (top) and bacteriorhodopsin modified
ders of magnitude longer wavelength would re- via single amino acid replacement (bottom; D96N mu-
tation). The contributions of water (dotted line) are high-
quire prohibitively large electromechanical dri- lighted by dividing the spectrum calculated for the pro-
ving of the birefringent material. We are exploring tein in the presence of water by that calculated in the
several means of generating alternating circular absence of water. Both macromolecules absorb strongly
polarization that appear well suited to the tera- and broadly across the terahertz, suggesting that terahertz
spectroscopy would be a relatively broad probe of macro-
hertz regime (Fig. 5). Each has unique advantages molecules in general. Of note, however, the distinct dif-
and disadvantages regarding its modulation rate ferences between the spectrum of the WT and mutant pro-
and its ability to generate pure, stable circular po- teins indicate that the precise details of the spectra depend
larization across a broad swath of the terahertz on the precise details of the molecular structure. Also
noteworthy is the observation that water enhances the in-
spectrum. tensity of all bands, and has a significant effect on the low-
Our first approach (Fig. 5, top) involves a rotat- est-frequency modes.
ing quartz wave plate. This consists of an x-cut (cut
parallel to the growth, or z-axis) quartz crystal,
which exhibits “slow” and “fast” axes that result 245° with respect to the wire grid and thus gen-
in a 1/4 l phase shift between vertically and hor- erating alternating circular polarizations.
izontally polarized light. A linearly polarized in- Our third approach (Fig. 5, bottom) uses a po-
put beam is passed through the plate, generating larizing Michaelson interferometer. Wire grid po-
an alternating right-circular/linear/left-circular/ larizers in each arm are orthogonally oriented. As
linear polarized output as the quartz is rotated. the length of one arm is changed, the phase rela-
Our second approach (Fig. 5, middle) utilizes tionship between vertical and horizontal compo-
a reflective circular polarizer, consisting of a free- nents defines a sequence of polarization states in-
standing wire grid placed 1/8 l in front of a mir- cluding pure left and pure right circular. The
ror. Half of the radiation is immediately reflected mirror is stepped by 1/4 wavelength to sequen-
by the grid, while the other half, in the orthogo- tially alternate between left and right circular for
nal polarization, is reflected by the mirror and de- each FEL pulse. This approach is especially at-
layed by 1/4 l resulting in a conversion from lin- tractive for continuous sources. Another signifi-
ear to circular polarization. A rotating polarizer cant advantage of this approach is that both am-
modulates the incident radiation falling on the plitude and phase can be adjusted in order to
circular polarizer, alternating between 145° and provide near-perfect circular polarization.
TCD FOR SPECTROSCOPIC LIFE DETECTION 495

Samples: the first control experiments. In order to ject (e.g., Yee et al., 1994; Maritan et al., 2000;
test and debug our initial TCD spectrometer Stasiak and Maddocks, 2000).
schemes, we have employed small, hand-wound Evolution, a critical component of the defini-
springs of 0.23 mm diameter made from 0.025- tion of life, will produce selective pressures that
mm copper wire. These materials are both con- ensure that these chiral, biologically produced
ducting and chiral (helical) on dimensions simi- macromolecules will be produced in enantiomer-
lar to those of the wavelengths being probed and ically pure (only one stereoisomer) form. This is
should exhibit very strong CD signals. Because because, by analogy to the observation that your
these materials are certain to exhibit strong CD, right shoe does not fit on your left foot, in gen-
we have employed “single-handed” samples of eral only one of the two enantiomers will be bio-
such springs, in random orientations, in our pos- logically active. The production of the incorrect,
itive control experiments. inactive enantiomer is thus almost always waste-
ful, and its elimination will be the subject of se-
lective pressures. [Pursuing the above analogy, if
evolution had invented footwear, there would be
RESULTS
selective pressure to suppress the asymmetry be-
Arguments, simulation results, and limited ex- tween our feet in order to avoid wasting the re-
perimental data supporting the concept that TCD sources required to maintain separate pathways
is an unambiguous and universal approach to life for the synthesis of both left and right shoes. In
detection are provided in the following sections, molecular systems such asymmetries cannot be
as well as a description of progress towards the suppressed—chirality is a fundamental charac-
building of the first TCD spectrometers. teristic of any intermolecular interaction that uti-
lizes three or more “attachment points” for recog-
nition (Ogston, 1948)—and thus, at the molecular
Key assumptions
level, the inevitable outcome of this selection is
Enantiomerically pure, chiral macromolecules: a homochirality rather than achirality.] Terrestri-
universal signature of life. All living systems, irre- ally, the symmetry between right- and left-
spective of their genesis, will employ macromol- handed macromolecules is perfectly and univer-
ecular “machines” in order to carry out their sally broken by the use of homochiral monomers
metabolic and reproductive functions. A critical in the synthesis of proteins, DNA, RNA, and
component of life, which is defined here as a self- polysaccharides. (Polymers composed of achiral
replicating chemical system capable of evolving, monomers also fold into chiral objects but, in con-
is the genetic storage of information (DNA and, trast, exhibit no net chirality because they popu-
to a lesser extent, RNA are the terrestrial analogs). late both enantiomers equally.) Chiral, macro-
This genetic material must encode the molecular molecular machinery should thus provide a
machines that are required to copy itself using as universal signature of chemical life, irrespective
building blocks only raw materials available from of its genesis.
the environment (with proteins and, to a lesser How common are chiral macromolecules in liv-
extent, RNA playing this role terrestrially). These ing organisms? We cannot, of course, know the
machines, like all “tools,” are fundamentally three- ultimate limits on this value, but considering the
dimensional objects. Moreover, like the vast ma- numbers for a typical terrestrial example would
jority of three-dimensional objects, these macro- seem a reasonable first approximation. The com-
molecular machines are chiral. That is, they lack mon bacterium Escherichia coli is composed of, by
any mirror planes of symmetry and thus are not mass, 70% water, 15% protein, 7% RNA and DNA,
superimposable onto their mirror images. In par- 3% polysaccharides, 2% lipids, and 3% inorganic
ticular, the most obvious chiral symmetry in ter- ions and small molecule metabolites (Ingraham et
restrial biomaterials arises due to the helical al., 1983). Of note, about 5% of the water is phys-
substructures that, despite their vastly differing ically coupled to chiral macromolecules in the
chemistries, are adopted by proteins, DNA, RNA, form of an inner hydration shell, and our calcu-
and polysaccharides (e.g., Fig. 1). It has been ar- lations indicate that this material behaves as if it
gued that the ubiquity of these helical structures were part of the macromolecule (see below). Thus
reflects unavoidable geometric constraints asso- approximately 30% of the mass of a typical ter-
ciated with packing a polymer into a compact ob- restrial cell is composed of chiral macromolecules
496 XU ET AL.

or is chiral by close physical association with such text (e.g., Pizzarello and Cronin, 2000), though
macromolecules. Given the abundance of chiral the mechanisms underlying these minor excesses
macromolecules and their argued association have not been identified. In contrast, it is well es-
with life irrespective of its chemical genesis, the tablished that minor enantiomeric excesses such
important remaining questions are whether chi- as these are insufficient to generate the homochi-
ral macromolecules are an unambiguous signa- ral, folded heteropolymers associated with life (e.g.,
ture of life and whether there exists a feasible, un- Bonner, 1995; Brandon and Tooze, 1999). It ap-
biased means of detecting them. pears, instead, that planetary-scale homochiral-
ity—net chirality of sufficient magnitude to gen-
Chiral macromolecules are an unambiguous signa- erate chiral macromolecules—can only occur
ture of life. It is widely held that enantiomerically when small-scale chiral fluctuations (randomly
pure (i.e., net chiral) materials unambiguously generated or biased by mechanisms such as those
distinguish biological material from abiological described above) are amplified via evolutionary
material (e.g., MacDermott et al., 1996; MacDer- selection. For this reason we, and many others
mott, 1997). And while most of the support for (e.g., MacDermott et al., 1996; MacDermott, 1997),
this assertion is, in a sense, negative observations, believe that the observation of net chirality is an
the large body of these observations argues unambiguous signature of biological processes.
strongly that the postulate is well founded. For
example, motivated by the observation that all Chiral macromolecules exhibit TCD. Extensive
terrestrial organisms use the L enantiomers of the simulations, theory, and empirical observations
amino acids and the D enantiomers of ribose or suggest that all polar macromolecules, irrespec-
deoxyribose in RNA and DNA, many creative tive of their chemistry, will exhibit collective vi-
theories have been explored in an effort to explain brational modes that are terahertz-active. If the
these homochiral “choices” in terms of enan- macromolecules are net chiral, these modes will
tiomeric biases predating the origins of life. Hy- produce TCD. The significant question remains,
pothesized mechanisms range from stereoselec- however, as to whether the TCD signals thus pro-
tive destruction of small molecules by circularly duced intense enough to be readily measured.
polarized UV light from magnetic neutron stars Here we explore these issues with reference to the
(reviewed by Bailey, 2000; Jorissen and Cerf, literature and to simulations.
2002), through the influence of parity violation in
the weak nuclear force (reviewed by Podlech, All polar macromolecules absorb strongly in the ter-
2001), to the influence of the “handedness” of the ahertz. Extensive molecular dynamics simulations
Earth’s orbit around the sun (e.g., He et al., 2000). (e.g., Brooks and Karplus, 1985, Hinsen, 1998; van
Despite serious effort, however, only the first of Vlijmen and Karplus, 1999; Hinsen et al., 2000,
these mechanisms has ever been reported to pro- Tama et al., 2000), normal mode calculations (e.g.,
duce any net chirality in the laboratory, and even Chen and Prohofsky, 1995; de Groot et al., 1998;
then the enantiomeric excess never exceeds a few Hery et al., 1998; Buck and Karplus, 1999), and
percent under even the most extreme experi- more limited experimental studies (e.g., Markelz
mental conditions (e.g., Bailey, 2000; Keszthelyi, et al., 2000; Globus et al., 2002; Woolard et al., 2002)
2001; Podlech, 2001; Wang and Liang, 2001). And indicate that effectively all biomolecules of .500
while beaker-scale syntheses have been reported atoms absorb strongly in the terahertz because of
that employ vigorous stirring (e.g., Kondepudi collective vibrational modes. It is this combina-
and Sabanayagam, 1994) to generate net chirality tion of broad absorption at terahertz frequencies
(apparently by crushing and mixing the first nu- and the potential for attendant broad CD (see be-
cleating crystal and thus ensuring that all subse- low) occurring almost irrespective of the chem-
quent crystals nucleate with the same handed- istry of the biomolecule that makes the terahertz
ness), either enantiomer is equally likely to be part of the spectrum attractive as an unbiased (in
produced in any given repeat of the experiment, terms of the precise chemistry of the molecular
and thus this effect is extremely unlikely to gen- machinery involved) vehicle for life detection.
erate net chirality on a planetary scale (reviewed
by Podlech, 2001). That said, a minor (2–9%), pre- All chiral macromolecules will exhibit CD at tera-
sumably abiologically produced enantiomeric ex- hertz frequencies. CD, which is the wavelength-
cess has been observed in an astronomical con- specific differential absorption of left and right
TCD FOR SPECTROSCOPIC LIFE DETECTION 497

FIG. 5. Schematics and images of TCD spectrometers built using a quartz 1/4 wave plate (top), rotating polariz-
ers (middle), and moving mirrors (bottom).
498 XU ET AL.

circularly polarized light, provides information produce TCD. The question remains, however, as
on the asymmetry of chromophores. The optical to whether this TCD is intense enough that it can
(UV, visible, near-infrared) chromophores of the be readily measured. A simple argument sug-
first row elements (i.e., elements that make strong gesting that the magnitude of TCD may be rela-
covalent bonds and are likely components of life: tively large is the observation that, in the tera-
carbon, oxygen, etc.), however, are achiral (e.g., hertz, the “chromophores” are the inherently
carbonyls, carbon–carbon double bonds, etc.) and asymmetric macrovibrational modes of chiral ob-
thus do not inherently exhibit CD. When they are jects. At optical wavelengths, where the vast ma-
present in a chiral, structured biomolecule, how- jority of likely chromophores are inherently sym-
ever, they are typically placed into asymmetric metric, the rare occurrence of inherent asymmetry
environments or participate in asymmetric exci- leads to 1–2 orders of magnitude larger CD in-
ton interactions and thus exhibit CD. Because op- tensities; for example, the inherently asymmetric
tical CD provides information on the structure of hydrocarbon hexahelicene exhibits a De/e ap-
biopolymers, the technique is an integral part of proaching 1% (Newman et al., 1967).
contemporary biophysics, and numerous, excel-
lent turnkey instruments are commercially avail-
Simulation results
able (reviewed by Kliger et al., 1990).
While optical CD is an extremely well-estab- We have attempted to develop more quantita-
lished technique, the approach has several draw- tive insights into the nature and magnitude of
backs as a general life-detection scheme. For ex- biomolecular TCD signals by computing the TCD
ample, the use of optical CD assumes that the spectra of two simple, computational models.
putative biomaterial in question contains strong Both models suggest that macromolecular vibra-
optical chromophores. While such chromophores tions of the helical structures inside typical bio-
are relatively common, the assumption that all molecules will lead to reasonably strong TCD,
life forms will utilize heteropolymers absorbing with precise spectral signatures that are a sensi-
at optical wavelengths should be avoided if pos- tive function of the distribution of mass, charge,
sible. A related concern regarding optical CD is and elastic couplings within the structure.
that by far the most likely chromophores in bio-
logical materials are composed of the cosmolog- Mass and spring model. CD at terahertz fre-
ically abundant, strongly covalent first row ele- quencies involves a very different set of excita-
ments hydrogen, carbon, nitrogen, and oxygen, tions than those responsible for UV, visible, and
and the optical chromophores of these structures near-infrared CD. UV CD and visible light CD are
generally produce only weak CD at optical wave- intimately related to electronic excitations. Near-
lengths. In the UV-visible spectral region, by far infrared CD is largely connected to vibrations as-
the most likely chromophores are electronic tran- sociated with the stretching and bending of bonds
sitions of carbon-, nitrogen-, and/or oxygen- (Deutsche and Moscowitz, 1968, 1970; Schellman,
containing double bonds. Double bonds of first 1973). But excitations in the terahertz and associ-
row elements are inherently symmetric struc- ated CD are best described as macromolecular vi-
tures, and thus they generally exhibit CD only as brations involving large pieces of the molecule
a second-order effect when they are placed in beating against each other (Fig. 1). In order to sim-
asymmetric environments or exhibit asymmetric ulate such motions, and the TCD spectral features
exciton interactions with other chromophores. associated with them, we have developed and
These second-order CD effects produce De/e of characterized a simple “mass and spring” model
typically 1023–1024 (e.g., Sprecher and Johnson, of a typical biopolymer helix. The torsional
1977). In the near-infrared, where likely chrom- acoustic modes supported by our specific model
phores are bond vibrations, the chiral asymmetry of the helix are shown (Fig. 3). As in linear chain
is even less intrinsic to the system, leading to De/e models of acoustic modes in solids, the highest-
of 1024 –1025 for both proteins (e.g., Pancoska et frequency mode corresponds to an acoustic
al., 1989) and DNA (e.g., Keiderling et al., 1989). wavelength of lmax 5 2a, twice the site spacing.
Because all macromolecules containing charges This particular calculation assumes that the helix
or polarized bonds absorb in the terahertz, and is infinitely long. A finite helix will support ad-
all chiral macromolecules will exhibit CD, almost ditional standing waves with wavelengths deter-
all biologically produced macromolecules will mined by the boundary conditions at the ends. If,
TCD FOR SPECTROSCOPIC LIFE DETECTION 499

guided by typical terrestrial examples (e.g., Fig.

1), we take the helix to be 7 periods long we
estimate that the longest wavelength is approxi-
mately lmax 5 140a, corresponding to a fre-
quency of ,0.5 THz.
In reality, the excitation of the helix in the bio-
molecule is not uniform; there are a variety of
masses, extraneous coupling to the environment,
different charges, and different local field correc-
tions. The electromagnetic field, very nearly uni-
form on the length scale of the helix, drives a
variety of modes determined by the Fourier trans-
form of the charge coupling parameters along the
helix. Here we have simply lumped a charge
every 14 sites to simulate a very nonuniform ex-
citation of the helix. The consequences of this par-
ticular distribution of charge are shown (Fig. 3).
The electromagnetic field at a given frequency
will resonate with a torsional acoustic mode at a
wavevector determined by the dispersion rela-
tion, but produces an amplitude exhibiting reso-
nant behavior at acoustic wavelengths or
wavevectors determined by the spatial variation
of the charge in the helix. We conclude that a par-
ticular helix will have a signature determined by,
among other things, the distributions of mass and
charge.
We have calculated the polarization produced
by a linearly polarized electromagnetic wave and
find that in addition to the polarization in the di-
rection of the applied terahertz electric field, the
system produces polarization perpendicular to it
and with the symmetry properties (Fowles, 1989)
required to produce rotation or TCD. Several rel-
evant features are observed in the resulting reso-
nant absorption and resonant TCD. As expected,
the helical model absorbs strongly at the fre-
quencies corresponding to the resonant excitation
shown (Fig. 3, middle). For the model density, the
absorption is quite strong. Indeed, it is well
known that glasses with reasonable charge den-
sity, such as sodium silicate glasses, exhibit
strong terahertz absorption that strengthens with
increasing frequency. The TCD spectral features
consist of a series of zero crossings (Fig. 3, bot-
tom). In the simple model the resonant features
are simply determined by the distribution of FIG. 6. Top: Simulation of the CD spectrum of an arti-
ficial bacteriorhodopsin-like system containing seven par-
charges. We may expect that any particular heli- tially charged point masses connected via weak harmonic
cal unit exhibits a unique, broad band “signature” bonds (see text). This artificial chiral system generates 15
pattern of such zero crossings. vibrational modes, the first three of which are illustrated
CD is always a small fraction of the direct ab- (lower panels). Electrostatic contours show the partial
charges on the point masses and the arrows indicate rel-
sorption. Using visible, UV, or infrared ratios as ative motion of the masses during the vibration. The ro-
a guide we would expect that the ratio will be tatory strengths were calculated using the VCD engine
within Gaussian-98 (Frisch et al., 1998).
500 XU ET AL.

,a/lTHz, where a is of the order of the atom or modes that characterize the terahertz vibrations
ion spacing. This is a very small number at tera- in bacteriorhodopsin. The fact that such modes
hertz frequencies. But the molecular unit, the consistently yield intense TCD bands is logical
“terahertz chromophore,” that is responsible for because the charged unit is vibrating within a chi-
the terahertz absorption involves many atoms ral arrangement. Based on these simulations we
and ions (Holzwarth and Chabay, 1972). For a he- propose that a majority of the strong protein vi-
lix 7 periods in length, the long wavelength brational modes below 3 THz will have observ-
acoustic mode in our simple model is ,140a, and able TCD activity with rotatory strengths in the
we expect to recover CD to absorption ratios that range 10242 –102 41 esu2 cm2 (102 65 –102 64 C2 m2).
are reasonable relative to those of electronic ex- However, the fact that individual modes have
citations or near-infrared vibrations. For this par- TCD activity does not guarantee that the net TCD
ticular, rather simple model we find De/e ,1024 , spectrum of the biomolecule will be intense. This
suggesting that TCD may be at least as intense as observation follows from the fact that two nearby
CD at optical wavelengths. We thus conclude that modes can have similar but opposite rotational
the collective vibrations of the helical structures strengths (e.g., modes 2 and 3 in Fig. 6). As the
inside typical biomolecules are likely to lead to density of vibrational modes increases, the prob-
reasonably strong and complex TCD spectra. ability of accidental cancellations of net rotational
strength will increase. Our simulations indicate
Normal mode analysis. We have also performed that the density of modes increases as the vibra-
more detailed calculations to model a specific, tional energy increases, and it is possible that
representative terrestrial biopolymer, the pro- large proteins may yield such a high density of
tein bacteriorhodopsin (Fig. 1). Because bacteri- TCD modes above 2 THz that the average rota-
orhodopsin, an archeal light-transducing protein tional strength will start to decrease at energies
(Oesterhelt and Stoeckenius, 1971), is thought to near or above this frequency. A definitive answer
have been an important contributor to terrestrial to this hypothesis is beyond the capability of our
life for nearly 3.5 billion years it is perhaps a par- current models and thus provides further impe-
ticularly appropriate choice for this study (Stuart tus for experimental studies of TCD.
and Birge, 1996). The Archaea from which it is
obtained, Halobacterium salinarum, is an extreme “Induced chirality” in macromolecular hydration
halophile that lives in natural environments such shells. We have also investigated the contributions
as the Dead Sea, the Great Salt Lake, or salt flats of hydration shells to likely biomolecular TCD
where the salt concentration is above that found signatures. Although water is the principal con-
in the oceans (as high as 25% NaCl). The deep stituent of biological systems, it is achiral, and
purple color of many salt flats is due to high con- thus bulk water will not exhibit TCD. Approxi-
centrations of this protein in the membrane of mately 1–5% of the water in a cell, however, is
these organisms. “biological water” that is in direct association
As described above, we have simulated the with polar or charged groups on the surface or
low-frequency vibrational modes of bacteri- inside of proteins and nucleic acids and may, due
orhodopsin. There are a total of 268 calculated to this “induced chirality,” be CD-active. In order
modes below 3 THz, roughly 20% of which are to test this, we have carried out a series of calcu-
strongly allowed. The most intense modes in- lations on biological water using Hartree–Fock
volve concerted motions of entire helices, a rep- and density functional (B3LYP) methods. We
resentative example of which is illustrated (Fig. were surprised to find that, by weight, biological
1). We simulate the TCD activity of these modes water will likely be the single most important
using charged heavy masses to simulate entire source of a TCD signature of a hydrated biolog-
helices connected by harmonic springs (Fig. 6). ical system. We find two sources of contribution.
Only some of the resultant modes are reflective The first is solvation enhancement. When water
of the modes observed in a protein. For example, is added to a system, it stabilizes ionic species
the lowest-frequency mode (mode 1) is an out-of- through direct association with the polar and
plane distortion that has no direct analogy in the charged amino acids. When these residues par-
bands calculated for bacteriorhodopsin. In con- ticipate in low-frequency, large-amplitude vibra-
trast, modes 2 and 3 of our simulation have much tional motion, the hydration shell moves to main-
in common with the in-plane helix translocation tain stabilization of the polar or charged species.
TCD FOR SPECTROSCOPIC LIFE DETECTION 501

The net result is a lowering of the frequency and terials, it is sufficient to detect the TCD arising
an enhancement in the intensity of the signal. The from small metal springs, which are both con-
frequency is lowered because the mass of the vi- ducting and chiral on the length scale of the ra-
brating system has increased. The intensity in- diation and thus exhibit strong CD (Fig. 7). We
creases because the magnitude of the charges is anticipate that, with the inclusion of reference de-
enhanced through solvation. Because a chiral tectors, antireflective coatings, significant, auto-
subsystem imparts chirality on the supersystem, mated signal averaging, and, ultimately, syn-
the first hydration shell enhances the chirality of chronous detection, it will be possible to improve
the local protein environment. The second hy- the signal-to-noise of this spectrometer to the
dration shell tends to slightly diminish the chi- ,105 we estimate would be suitable for a life-
rality because of fluid motion, and subsequent detection approach.
shells have relatively little impact. In all cases we
investigated, however, water enhanced the TCD
signature. We also found that there were some DISCUSSION
specific cases of very intense TCD signals associ-
ated with water “wires” that bridged the gap be- Here we have argued that, irrespective of the
tween two charged residues. In the five examples chemistry upon which they are based, all forms
studied, water wires containing from three to of chemical life will employ enantiomerically
seven molecules exhibited low-frequency modes pure, chiral macromolecules as the “machines”
in the 0.3–3 THz region, and many of these modes employed in their metabolism and reproduction.
had rotatory strengths above 10242 esu2 cm2 . The results of our simulations strongly suggest
From a mass-weighted perspective, water wires that all such macromolecules will absorb strongly
between charged residues were the single most in the terahertz, as has been previously docu-
important source of TCD bands that we observed mented experimentally for terrestrial biopoly-
in our simulations. mers, and that these absorbance features will ex-
hibit relatively strong CD. There appear to be
Development of TCD spectrometers
Previous attempts to build TCD spectrometers
have met with only limited success (Polavarapu
and Chen, 1994; Polavarapu and Deng, 1996). We
are currently developing three competing TCD
spectrometer schemes (Fig. 5) that potentially of-
fer significant advantages over previous efforts in
terms of both signal-to-noise ratios and possible
systematic errors. The three approaches differ in
the manner in which alternating circular polar-
ization is generated.

TCD appears technologically feasible. To date the

most progress has been made with a spectrome-
ter that generates alternative polarizations via a
quartz 1/4 l wave plate. Because quartz crystals
are birefringent, the indices of refraction for ver-
tically and horizontally polarized light differ at FIG. 7. TCD signals of 0.23-mm-diameter, right-
terahertz wavelengths, an effect that can be used handed, copper springs observed at 0.14 THz using the
rotating quartz 1/4 waveplate spectrometer. The repro-
to construct a 1/4 l wave plate that generates al-
ducibility of measurements, collected at 180° intervals,
ternating right- and left-circularly polarized light clearly demonstrates the impressive signal-to-noise ob-
as the quartz is rotated (Fig. 5, top panels). The tained with this spectrometer despite the relatively minor
signal-to-noise ratio of the currently imple- averaging used in this data collection (each point repre-
sents eight replicates). The non-equivalence of the ob-
mented (manually operated and thus with lim-
served transmittance of linearly polarized radiation at 90°
ited averaging) spectrometer is ,102. While this intervals reflects the small linear dichroism associated
is insufficient to detect TCD in terrestrial bioma- with these (imperfectly helical) springs.
502 XU ET AL.

several possible means of building a spectrome- X-ray and NMR spectroscopy. J. Am. Chem. Soc. 121,
ter to detect this TCD, one of which we have built 9645–9658.
and used to have measure the TCD of a macro- Cheeseman, J.R., Frisch, M.J., Devlin, F.J., and Stephens,
P.J. (1996) Ab initio calculation of atomic axial tensors
scopic, positive control. The TCD life-detection
and vibrational rotational strengths using density func-
approach not only provides a potential test for tional theory. Chem. Phys. Lett. 252, 211–220.
chirality as a signature of life in unknown, com- Chen, Y.Z. and Prohofsky, E.W. (1995) Sequence and tem-
plex materials, but also serves to highlight the perature dependence of the interbase hydrogen-bond
much broader and hitherto poorly explored con- breathing modes in B-DNA polymers. Comparison
cept of detecting life via the macromolecular ma- with low-frequency Raman peaks and their role in he-
chinery with which it carries out its metabolic and lix melting. Biopolymers 35, 573–582.
de Groot, B.L., Hayward, S., van Aalten, D.M.F., Amadei,
genetic functions.
A., and Berendsen, H.J.C. (1998) Domain motions in
bacteriophage T4 lysozyme: a comparison between
molecular dynamics and crystallographic data. Proteins:
ACKNOWLEDGMENTS Struct. Func. Gen. 31, 116–127.
Deutsche, C.W. and Moscowitz, A. (1968) Optical activ-
Funding for the University of California, Santa ity of vibrational origin. I. A model helical polymer. J.
Chem. Phys. 49, 3257–3272.
Barbara portions of this research program were
Deutsche, C.W. and Moscowitz, A. (1970) Optical activ-
provided by NASA (NAG5-12150) and the ARO ity of vibrational origin. II. Consequences of polymer
(DAAD19-02-01-0080), and for the University of conformation. J. Chem. Phys. 53, 2630–2635.
Connecticut group from the NIH (GM-34548) and Fowles, G.R. (1989) Introduction to Modern Optics, 2nd
NSF (EIA-0129731). edit., Dover Publications, New York.
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E.,
Robb, M.A., Cheeseman, J.R., Zakrzewski, V.G., Mont-
gomery, J.A., Jr., Stratmann, R.E., Burant, J.C., Dap-
ABBREVIATIONS prich, S., Millam, J.M., Daniels, A.D., Kudin, K.N.,
Strain, M.C., Farkas, O., Tomasi, J., Barone, V., Cossi,
CD, circular dichroism; CW, continuous wave; M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C.,
PAH, polycyclic aromatic hydrocarbon; TCD, ter- Clifford, S., Ochterski, J., Petersson, G.A., Ayala, P. Y.,
ahertz circular dichroism; WT, wild type. Cui, Q., Morokuma, K., Malick, D.K., Rabuck, A.D.,
Raghavachari, K., Foresman, J.B., Cioslowki, J., Ortiz,
J.V., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P.,
Komaromi, I., Gomperts, R., Martin, R.L., Fox, D.J.,
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