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ANALYTICAL CURRENTS A bright idea for antigen detection Geoffrey Waldo, Andrew Bradbury, and colleagues at the Los Alamos National Laboratory, Baylor College of Medicine, and Lewis and Clark College have generated libraries of molecules that combine the specificity and affinity of antibodies with the real-time fluorescence of green fluorescent protein (GFP). The new molecules, called fluorobodies, exhibit many of the same properties as antibodies without the need for detection with labeled secondary antibodies. Previous attempts by other researchers to insert sequences into GFPs yielded molecules with severely compromised fluorescence. Thus, Waldo, Bradbury, and colleagues made insertions into a highly stable mutant form of GFP, reasoning that a more stable starting molecule would tolerate insertions better than the normal form. To get GFPs that would functionally mimic antibodies, the researchers placed looped antigen-binding
regions of antibodies into the loops of GFP molecules. Fluorobodies were selected by phage display, wherein one fluorobody is attached to a phage that infects bacteria. Sixtyfive percent of the bacterial clones were fluorescent, which indicates that the fluorobodies within the clones retained this property of GFP. The researchers screened the new molecules against five different antigens and found that ~20–80% of the fluorobodies tested specifically bound a particular antigen. Green bacterial colonies contain functional fluoroThose fluorobodies that were bodies. (Adapted with permission. Copyright 2003 fluorescent and specific were com- Nature Publishing Group.) pared to antibodies in a number of the researchers conclude that the fluotests, including flow cytometry, gel shift robodies function in a way similar to assays, protein arrays, and immunofluoantibodies. (Nat. Biotechnol. 2003, 21, rescent staining of cells. On the basis of 1473–1479) the data generated by these experiments,
An explosives detector with a bang J. D. Adams, T. Thundat, and co-workers at
TNT molecules easily stick to the un-
TNT
the Oak Ridge National Laboratory, the Uni-
coated cantilevers of the new device.
versity of Tennessee, the U.S. Department
As the molecules slowly desorb, the re-
of Homeland Security, and the University of
searchers apply a voltage to a piezoresis-
Nevada have developed a trinitrotoluene
tor in the cantilever, which increases its
(TNT) microsensor that is much smaller
temperature and causes a miniature explo-
than the bulky ion mobility spectrometers
sion. The cantilevers are robust and func-
(IMSs) currently used in airports. Although
tion normally after hundreds of explosions.
the sensor is still not optimized, it has a 11
Piezoresistive heater
The researchers also tested other sub-
100 M
TNT microsensor. (Adapted with permission. Copyright 2003 Nature Publishing Group.) was applied. The authors say that they have
detection resolution of 70 pg (1.9 10
stances that they thought might interfere
detected additional explosives with the de-
molecules) TNT, which researchers say is
with detection of TNT, such as water, ace-
vice that detonate at different deflagration
comparable to that of the most recently
tone, ethanol, and gasoline, but all of these
points and desorption times. (Nature 2003,
reported IMS.
desorbed before the deflagration voltage
425, 474)
© 2004 AMERICAN CHEMICAL SOCIETY
J A N U A R Y 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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news
ANALYTICAL CURRENTS
Water-soluble Hg(II) sensor The sensor re-
of the Massachusetts Institute of Technolo-
sponse is not affected
gy have developed a fluorescent sensor
by the presence of
that is capable of detecting environmentally
most metal cations
relevant concentrations of Hg(II) in water.
found in the environ-
The sensor is highly selective for Hg(II),
ment, with the excep-
even in the presence of several other met-
tion of Cu(II). In addi-
als commonly found in the environment.
tion, the binding of
Unlike most other small-molecule Hg(II)
Hg(II) to the sensor
probes, it does not require the use of or-
molecule is reversible.
ganic solvents.
When the researchers
30 Fluorescence intensity
Stephen J. Lippard and Elizabeth M. Nolan
25 20 +Hg(II)
15 10 5 0 480
500
520 540 560 580 Wavelength (nm)
600
620
Fluorescence response of the mercuric ion sensor to the addition of Hg(II) in water at neutral pH and ionic strength of 100 mM. The concentration of the sensor molecule is 1 µM.
The new water-soluble sensor uses
added a heavy metal
fluorescein as the reporting group, and
ion chelator to a mix-
because Hg(II) ions have a high affinity for
ture containing the sen-
sulfur, a sulfur-containing moiety is incor-
sor molecule and Hg(II), they saw an imme-
set the standard for the maximum allowable
porated into the ligand framework. The un-
diate reduction in fluorescence to within
concentration of Hg(II) in drinking water at
bound sensor exhibits an emission maxi-
20% of the background level. Subsequent
2 ppb. When the researchers added the
mum at 524 nm and a low quantum yield of
addition of Hg(II) restored the fluorescence
new sensor to an aqueous solution contain-
0.04. When Hg(II) binds to the sensor, the
because of Hg(II) complexation.
ing 2 ppb Hg(II), they observed a fluores-
emission maximum red shifts to 528 nm and
Hg(II) is converted by bacteria into toxic
cence increase of about 11%, which is an
the quantum yield increases to 0.11. The re-
methylmercury in the marine environment,
indication that the sensor can detect Hg(II)
sulting enhancement in fluorescence inten-
where it bioaccumulates up the food chain,
at the low levels typically found in environ-
sity is due to the formation of a 1:1 complex
particularly affecting large edible fish. The
mental matrixes. (J. Am. Chem. Soc. 2003,
between Hg(II) and the sensor.
U.S. Environmental Protection Agency has
125, 14,270–14,271)
Photolabels to study Ras interactions Ras proteins—which are critical to cell growth and differentiation, the cell cycle, and apoptosis—need to be localized at the plasma membrane to work, and the posttranslational modifications that control this localization are still being studied. Now, Herbert Waldmann and his colleagues at the MaxPlanck-Institut für molekulare Physiologie and the Universität Dortmund (both in Germany) describe a “construction kit” for the modular assembly of “photoactivatable” Ras peptides to examine the protein–protein interac8 A
tions of this crucial signaling molecule. The new kit combines solid- and solution-phase syntheses to make peptides that incorporate benzophenone (BP) photolabels. BPs were chosen because they have previously been applied to protein detection, and their excitation wavelength is not harmful to proteins. The photolabel was placed within the farnesyl group because it is likely that this group is involved in membrane binding events. Different functional groups, such as biotin, can be incorporated at the N-terminal ends
A N A LY T I C A L C H E M I S T R Y / J A N U A R Y 1 , 2 0 0 4
of the peptides. In all, 24 peptides were synthesized. When two conjugates were attached to oncogenic N-RasG12V181 and the labeled proteins were inserted into cells, they retained their biological activity. In other experiments using photolabeled geranyl-BP-N-Ras and farnesyl-sensitive guanine nucleotide exchange factor, hSos1, the researchers looked at the role of the farnesyl group in Ras signaling, which is still being debated. (J. Am. Chem. Soc. 2003, 125, 12,749–12,758)
news
Budding lipid membrane research Lipid bilayer membranes are a cell’s shape shifters, morphing to form buds and splitting into vesicles. Theoretical descriptions of this behavior have been difficult to validate, but Tobias Baumgart and Watt Webb at Cornell University and Samuel Hess at the National Institute of Child Health and Human Development have developed an optical method to test current theories. These analyses have led to experimental estimates of the boundary tension, which is thought to control membrane deformation. The researchers studied giant unilamellar vesicles formed from a mixture of the lipids sphingomyelin, cholesterol, and dioleoylphosphatidylcholine (DOPC). The lipids separate into two different liquid phases: Sphingomyelin and cholesterol exist in a liquid phase with shortrange order, while DOPC prefers a disordered liquid phase. The two phases were labeled with different dyes and stud-
ied with two-photon microscopy to relate the composition of the phases, or domains, to the mechanical properties of the vesicles, such as line and lateral tensions. The researchers demonstrated that line tension drives vesicles toward a “limit shape”—a large sphere with semicircular protrusions. When temperature is increased or osmotic pressure changes, a bud may form abruptly, which suggests that an energy barrier was overcome. They also observed long-range ordering, which appeared as locally parallel stripes and almost soccer ball-shaped hexagonal arrays of circular domains. When temperatures were increased to just below the point at which homogeneous membranes formed, circular domains began to undulate laterally and assume elliptical shapes. At even higher temperatures, a “stripe out” would occur—a phenomenon whereby numerous thin stripes with varying lengths
Bioactive aerogels for sensing Because aerogels are so porous, incorpo-
They propose that the outer protein
rating active proteins into them ought to
layer is damaged and unfolds dur-
yield biosensors that respond and regener-
ing aerogel processing. However,
ate much faster than sol–gel ones. Unfortu-
this sacrificial “skin” protects the
nately, proteins generally do not survive the
proteins underneath, which bind
harsh processing required to make aero-
NO in the same fashion as they
gels. Now, Debra Rolison and colleagues at
do in solution.
the U.S. Naval Research Laboratory intro-
Rolison and her colleagues
duce a method for preserving the activity of
also believe that this process may
cytochrome c during the formation of an
help shed some light on how life
aerogel and thereby produce a NO detector.
evolved. According to one prebiot-
The new aerogel technique uses small
ic evolutionary theory, single cells
(a)
(b)
(c)
(d)
A bilayer composed of two lipid phases (red and blue). (a) Separation of the phases at room temperature. (b) Formation of a bud. (c) Long-range ordering. (d) Stripes and an elliptical shape. (Adapted with permission. Copyright 2003 Nature Publishing Group.)
undulate rapidly, similar to the rippling instability of collapsing bubbles. (Nature 2003, 425, 821–824)
Adsorbed monolayer of cyt. c
Buffer-like internal environment
Au colloid Damaged (unfolded) layer of cyt. c
SiO2 aerogel environment ~102 nm
A suggested model of a gold particle with layers of active cytochrome c surrounded by a damaged outer layer.
amounts of colloidal gold that act as
began to form when precellular
nanoparticle seeds for the formation of self-
macromolecules were stabilized in
organized protein–protein superstructures
porous minerals in undersea thermal vents.
molecules is still possible. The researchers
that are captured during gelation of a silica
This, the researchers argue, is analogous
further suggest that an outer shell of sacri-
sol. When the nanoparticles are present,
to their bioaerogel, because in both cases,
ficial macromolecules may have isolated
>80% of the cytochrome c molecules remain
biomolecular structures are isolated in a
the interior units from damage in a prebiot-
viable after processing, the researchers say.
porous architecture, yet transport of small
ic system. (Nano Lett. 2003, 3, 1463–1467)
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news
ANALYTICAL CURRENTS hv2
Quantum dots illuminate DNA activities Itamar Willner and colleagues at the Hebrew University of Jerusalem (Israel) examine the modification of DNA with telomerase and polymerase using fluorescence resonance energy transfer (FRET) between CdSe–ZnS quantum dots and a dye. The quantum dots’ surfaces are modified and attached to a segment of DNA, which is then incubated with the DNA modification enzyme and a mixture of dATP, dCTP, dGTP, and Texas-red 14dUTP. During the progression of the telomerization or polymerization, the fluorescence emission shifts as the 560nm emission from the quantum dots decreases and the 610-nm emission of the dye increases (using 400-nm excitation). In the absence of the enzymes, the emis-
(a)
hv1'
sions don’t change. Thus Telomerase the researchers conclude that dUTP + dNTPs hv1 FRET between the quantum hv2 dots and the dye is responsible DNA M 1 3 for the shift in emission. Kleno DNA hv1 w fra Telomeric DNA chains meagmen t dUTP sured with this technique and + dNT P s atomic force microscopy were (b) 1 nm high and 300 nm long, which corresponds to 1000 base hv1 units. Both telomerase and polymerase showed similar kinetics, Schematics of how fluorescence resonance energy reaching saturation after 60 min. transfer occurs between a quantum dot and dye The researchers suggest that incorporated into DNA during (a) telomerization and the technique may prove useful (b) polymerization. in the detection of malignant cence when and if hybridization and cells. It could also be applied to chipreplication proceed. (J. Am. Chem. Soc. based DNA sensors because it functions 2003, 125, 13,918–13,919) like a logic gate, only changing fluores-
Simple approach to IC50
P-pep
A remarkably simple NMR technique offers
mean that samples with numerous compo-
a rapid method for determining the inhibi-
nents present no problems. The protein
tion or activation of an enzyme—an impor-
concentration needed is determined by
tant factor in evaluating molecules as po-
the speed of the enzymatic reaction. sured as a function of the substrate. Alter-
19 strates with a –CF3 moiety and use F
natively, the speed of the reaction plotted
NMR to measure the binding constant
versus substrate concentration provides
(measured as KM) and the 50% mean inhi-
the same value. 19 A plot of the F signal of the starting
bition concentration (IC50) with protein con-
substrate or its product at various inhibitor
The approach is easily integrated into
concentrations against the inhibitor con-
high-throughput screening strategies.
centration yields the IC50. In the absence of
The new technique is called three
allosteric effects, a single point measure-
fluorine atoms for biochemical screening
ment yields an effective IC50. The authors
(3-FABS). By monitoring only the fluorine
report obtaining values in as little as 3 min
NMR spectrum, it offers a single, intense
(64 scans), which drops to a mere 12 s
peak to monitor; a chemical shift that is
when a cryoprobe is used. 3-FABS could also play a role in genomic
sensitive to minor structural changes; and
*
30 25 20
H89
15
IC50 = 0.72 0.05 M
10 5 0 0.1
centrations as low as a few nanomolars.
* 19F
[pep] in M
colleagues from Pharmacia (Italy) tag sub-
+ H89
pep
19 To determine KM, the F peak is mea-
tential drug leads. Claudio Dalvit and
+ 4 cpds + 4 cpds + H89
1
[H89] in M
10
Top: 19F NMR of substrate (pep) and the enzyme reaction product (P-pep) in the presence of four compounds and the inhibitor H89. The first and third spectra show product formation, which is halted whenever H89 is present. Bottom: Plot to determine IC50. test substrates can then be used to assign a
no spectral interferences. Even medium or
screening. A library of CF3-labeled substrates
protein or proteins of unknown function to
weak inhibitors can be detected, and clean
of known enzymes is created, and their rele-
a particular category, such as proteases.
spectra and low protein concentrations
vant chemical shifts are measured. These
(J. Am. Chem. Soc. 2003, 127, 14,620–14,625)
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