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ANALYTICAL CURRENTS How many antibodies work after QD conjugation? Chemical conjugation of antibodies to semiconductor quantum dots (QDs) is attractive because the proteins of interest can be visualized. But Gabriel Silva and colleagues at the University of California San Diego have found that fewer antibodies function correctly on the QDs than previously thought and that the number varies significantly for two different conjugation methods. Attaching an appropriate number of functional antibodies to the QDs is critical for the proper interpretation of data derived from biological samples. If the conjugation conditions aren’t optimized, the antibody–QD complexes may label proteins nonspecifically and yield reproducible artifacts. To determine the number of functional immunoglobulin G (IgG) antibodies that are conjugated to QDs, Silva and colleagues compared a commonly used direct covalent conjugation method based on a reduced disulfide maleimide reaction with a biotin–streptavidin method.
(a)
(b)
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µ mµm 10
(c)
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(a) Correct labeling of a cytoskeletal protein by antibody–QD complexes produced by the streptavidin–biotin method. Nonspecific staining occurred (b) when the same antibody was conjugated directly to the QDs and (c) when the QDs were functionalized with antibodies against a cell-surface protein not found in these cells.
The antibody–QD complexes were run on SDS-PAGE gels under conditions in which the functional component of the antibodies separated from the QDs. The investigators then transferred the antibody fragments onto a membrane to determine the identity and the amount of the antibodies. They quantitatively compared the amount of transferred antibody to the known antibody concentration used in the conjugation scheme to calculate the number of bound antibodies on the QDs.
The investigators found that the number of functional antibodies bound to QDs via direct conjugation was, on average, much less than one functional IgG molecule per QD (0.076 ± 0.014). Antibodies bound to QDs via the streptavidin–biotin system worked better, with 0.60 ± 0.14 IgG molecules per QD for a 1:1 IgG:QD molar ratio and 1.3 ± 0.35 for a 2:1 ratio. The approach is applicable to other types of functionalized nanoparticles. (Nano Lett. 2007, DOI 10.1021/nl062706i)
SERS at Pd and Pt surfaces Philip Bartlett and colleagues at the Univer-
bility and are widely used as electrodes and
remaining thin film is stippled with a regular
sity of Southampton (U.K.) have developed
catalysts. Efforts to fabricate SERS-active
array of void spaces. The thickness of the
a new method for the fabrication of sur-
Pd and Pt surfaces have been mildly suc-
film is controlled by varying the charge
face-enhanced Raman spectroscopy
cessful but often result in irregular surfaces
passed during electrodeposition. The re-
(SERS)-active Pd and Pt surfaces. SERS is
that unevenly enhance the Raman signal.
searchers note that SERS on their Pd and
typically limited to the roughened surfaces
Bartlett and co-workers fabricate their Pd
Pt surfaces is uniform across the entire ex-
of metals such as Ag, Au, and Cu, but SERS
and Pt surfaces by electrodeposition through
panse and that the surfaces can be cleaned
at Pd and Pt surfaces is desirable because
closely packed monolayers of polystyrene
and reused up to 6 months later. (J. Am.
these two metals have higher surface sta-
spheres. When the spheres are removed, the
Chem. Soc. 2007, 129, 7399–7406)
© 2007 AMERICAN CHEMICAL SOCIETY
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ANALYTICAL CURRENTS
How do constituents in plasma membranes move around? Many cell-signaling events start at the
membrane. Another model, called hop diffu-
120 nm, they improved the spatial and time
plasma membrane, where a sequence of
sion, involves transmembrane proteins in-
resolutions of state-of-the-art single-mole-
tightly regulated protein interactions trig-
teracting with the cytoskeleton sitting below
cule imaging devices. Then, the investiga-
gers the release of intracellular second
the plasma membrane. Evidence for the
tors measured the lateral movement of indi-
messengers. Researchers have been work-
model comes from single-particle tracking
vidual proteins on a millisecond timescale
ing to come up with a model of how the in-
experiments of gold-labeled phospholipids
with a positional accuracy of 22 nm.
teractions in the plasma membrane take
or lipid-anchored proteins. In these studies,
place. Now, Gerhard Schütz and colleagues
plasma-membrane constituents appear to
hop diffusion model, because Schütz and
at Johannes Kepler University Linz and the
be transiently confined in periodic corrals.
colleagues were unable to observe the
Medical University of Vienna (both in Aus-
Schütz and colleagues investigated the
The experimental data didn’t support the
transient corrals. The protein seemed to
tria) have disputed one of the suggested
mobility of a protein called CD59 in the plas-
freely diffuse by Brownian motion. The in-
mechanisms by carrying out single-mole-
ma membranes of live cells. They labeled
vestigators suggest that the gold labeling
cule fluorescence microscopy experiments.
the protein with fluorescent antibody frag-
causes exaggerated confinement of pro-
ments to ensure that the fragments didn’t
teins and lipids because of its ability to
Brownian motion, have been proposed to
hinder the protein’s movements. To detect
temporarily bind to other membrane pro-
describe the interactions in the plasma
the transient corrals at the reported size of
teins. (Biophys. J. 2007, 92, 3719–3728)
Several models, including lipid rafts and
Fast MS of viruses and cells Laser-induced acoustic desorption (LIAD) has recently allowed scientists to study intact bioparticles such as viruses, bacteria, and whole mammalian cells by MS and to distinguish among different populations of cells on the basis of the average mass. In LIAD, the m/z is determined by light-scattering measurements, but to determine the mass of the particle, the charge state must be changed several times and the light scattering remeasured. This process can take up to 30 minutes per particle; measuring the masses of a population of cells is thus impractical. Now, Chung-Hsuan Chen and colleagues at Academia Sinica (Taiwan) report a new method, charge-monitoring LIAD (CLIAD) MS, for a more rapid determination of bioparticle mass. In the new technique, the researchers eliminate the need for light-scattering
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measurements and, instead, use a charge-detection plate to measure the number of charges on a particle. In the past, direct charge measurement of bioparticles has been hampered by extremely high background noise. CLIAD solves this problem by increasing the number of charges on each particle with a mild corona discharge; this greatly enhances the S/N. The investigators estimate that CLIAD MS can analyze ~7200 particles per hour, an improvement of 3 orders of magnitude over LIAD MS. (Angew. Chem., Int. Ed. 2007, 46, 3865– 3869)
A N A LY T I C A L C H E M I S T R Y / A U G U S T 1 , 2 0 0 7
CCD Sample plate
Nd:YAG laser
Quadrupole ion trap Charge detector
HeNe laser
In CLIAD MS, the Nd:YAG laser induces cell desorption, and the HeNe laser illuminates the cells for detection by the CCD camera. The inset shows a CCD image of the trapped cells. (Adapted with permission. Copyright 2007 Wiley-VCH.)
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Shifting ideas about protein NMR In NMR protein structure determination, the modest chemical shift has long been either ignored or marginalized in favor of nuclear Overhauser enhancement or residual dipolar coupling data. Chemical shift data can be acquired quickly but have been difficult to inter-
Studying protein–DNA interactions inside porous vesicles Although the behavior of molecules can be observed inside vesicles, the vesicle’s lipid bilayer acts as a barrier to solutes. So Taekjip Ha and colleagues at the University of Illinois Urbana–Champaign have shown how buffer can be exchanged and molecular interactions inside vesicles can be manipulated. Defects in the lipid packing begin to appear when the temperature approaches the melting point of phospholipids. Previous studies showed that at room temperature, vesicles of dimyristoyl phosphatidylcholine (DMPC) have pores large enough to allow the exchange of adenosine diphosphate and ions but small enough to retain DNA.
pret in complex protein NMR spectra. Now, Michele Vendruscolo and colleagues at Cambridge University (U.K.) have developed a method to predict protein structural information from NMR chemical shifts alone. Their method, protein structure determination with chemical shift restraints (CHESHIRE), is a computational technique based on the molecular fragment replacement approach. CHESHIRE has three steps: 3PRED, in which the experimental chemical shifts are used to predict the secondary structure of small protein fragments; TOPOS, in which a library of trial conformations for each fragment is screened against a database to search for fragments with similar features; and molecular fragment replacement, in which the fragments are assembled and the structures are refined. The researchers tested their method on 11 proteins of up to 123 residues in length and found that their method predicted tertiary conformations very similar to high-resolution structures determined by other methods. (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9615–9620)
ATPgS replaces the ATP inside the vesicle and induces a change in the interaction between the RecA protein (blue) and the DNA (black with fluorescent dyes on the ends). (Adapted with permission. Copyright 2007 National Academy of Sciences, U.S.A.)
Ha and colleagues analyzed interactions between the E. coli RecA protein and DNA. RecA proteins bind to single-stranded DNA to form a filament. The investigators modified ends of single-stranded DNA with fluorescent dyes for single-molecule fluorescence resonance energy transfer (FRET) measurements and encapsulated the tagged DNA with RecA in DMPC vesicles. When the investigators added adenosine triphosphate (ATP), the vesicle pores permitted the exchange of ATP between the vesicle interior and the surrounding solution. RecA monomers dissociate from the filament upon ATP hydrolysis. The FRET efficiency fluctuated between two states—a high-efficiency state when the DNA wasn’t bound by RecA and a low-efficiency state when RecA assembled into filaments on the DNA. The investigators observed repetitive binding and dissociation of the same RecA filament on the DNA, and the rebinding rate was 2 orders of magnitude greater than when the experiment was done outside the confines of a vesicle. The results suggested a new nucleation pathway for RecA filaments. When a nonhydrolyzable form of ATP, ATPgS, was introduced, the FRET signal stayed at the lower state, indicating the stability of the filament. When ATP replaced ATPgS, the FRET signal resumed its fluctuations. The investigators say their method provides a way to study transient and weakly interacting biological complexes. (Proc. Natl. Acad. Sci. U.S.A. 2007, DOI 10.1073/pnas.0610673104)
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ANALYTICAL CURRENTS An NMR structure from micrograms of protein
GAETANO T. MONTELIONE
copy, and 3D 13C-edited nuclear OverGaetano Montelione and colleagues at To test the mass detection limits of Rutgers University, Bruker BioSpin hauser enhancement spectroscopy, to the microcoil probe, the researchers Corp., and the Robert Wood Johnson obtain structural information. For comused the 68-residue TRAM domain Medical School have reported the first parison, they obtained the same NMR protein from Methanosarcina mazei. complete NMR structure of data for 1600 µg of the
