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Following single-protein folding
New electrokinetic focusing method David Ross and colleagues at the Nation-
can support micelle formation. The re-
al Institute of Standards and Technology
searchers applied the new technique to
have created a new tool for the rapid,
focus the zwitterionic dye rhodamine B. A
sensitive, and selective separation of
concentrated band formed within seconds,
neutral and ionic hydrophobic analytes.
and a 13-fold increase in concentration oc-
Called micellar affinity gradient focusing
curred after 50 s. Similarly, they used MAGF
(MAGF), the technique combines micellar
to focus anthracene. In that case, the con-
electrokinetic chromatography (MEKC)
centration increased 27-fold in 30 s. Finally,
and temperature gradient focusing (TGF).
they successfully used MAGF to simultane-
MEKC separates neutral analytes on the
ously concentrate and separate two similar
basis of partitioning into an ionic micelle.
dyes—rhodamine B and rhodamine 110.
TGF separates species on the basis of dif-
With any separation method, there is a
ferences in electrophoretic mobility. When
tradeoff between sensitivity and resolution.
they are combined together as MAGF, the
However, with MAGF, the injection time is
two techniques are more sensitive than CE
also a factor. An improvement in detection
or MEKC alone. In addition, MAGF can be
limit therefore does not always mean poorer
readily implemented in a microfluidic device,
resolution. For example, if higher concen-
with shorter channels than CE or MEKC.
trations are needed, a longer injection time
MAGF is both a concentration and sep-
could be used without degrading the resolu-
aration method. The concentration or fo-
tion. The researchers are currently investi-
cusing mechanism involves creating a
gating the limits of this three-way tradeoff.
temperature gradient along the separation
(J. Am. Chem. Soc. 2004, 126, 1936–1937)
channel. The phase ratio (the ratio of the volume occupied by the micellar phase to
(a)
the volume of the mobile phase) decreas-
High retention
Low retention
+V
es with temperature. By establishing a Buffer Micelles Analyte
gradient in the phase ratio, a spatial gradient in the retention factor is created. The end result is a spatial velocity gradient,
(b)
0s
which allows the velocity of the analyte to
10 s
be zero at a particular point in the chan-
20 s
nel, where focusing and separation occur.
30 s
Ross and colleagues demonstrated MAGF by utilizing the surfactant sodium dodecyl sulfate, which is commonly used in MEKC, and a carbonate buffer. Unlike TGF, which requires a buffer whose ionic strength is dependent on temperature, MAGF can be performed in any buffer that
40 s 50 s
(a) Schematic of MAGF. (b) MAGF focusing of rhodamine B over time. Each image shows a 1.5-mm length of the separation channel.
Julio Fernandez and Hongbin Li of Columbia University have observed the complete folding course of a single protein. Their measurements show that protein folding occurs through a series of continuous stages, challenging the commonly held perception that protein folding consists of discrete, well-defined steps. Fernandez and Li used force-clamp atomic force microscopy (AFM) to monitor the end-to-end length of the small protein ubiquitin as it underwent the unfolded to folded transition. In particular, the investigators studied ubiquitin in the form of a polyprotein, in which nine ubiquitin molecules were arranged in tandem. In the force-clamp AFM experiments, the polyprotein was picked up by an AFM cantilever and unfolded by a 120-pN stretching force. The 120-pN stretching force was then released to 15 pN, and the folding of the polyprotein was monitored. To confirm the polyprotein had completed its folding course, the investigators reapplied the 120-pN stretching force to unravel the polyprotein again. Fernandez and Li performed 81 experiments and found that most of the folding trajectories of the ubiquitin polyprotein were qualitatively similar. But they were unable to observe identical sets of trajectories. The results suggest the existence of multiple folding pathways for ubiquitin. During the course of their experiments, the investigators found that the time taken for the protein to fold was dependent on the contour length of the unfolded protein and the applied stretching force. As the protein collapsed after stretching, Fernandez and Li found the end-to-end protein length fluctuated widely, but the fluctuations disappeared when the protein underwent its final folding contraction. These findings open up avenues to future investigations for a more detailed understanding of the physical basis of protein folding. (Science 2004, 303, 1674–78) M A 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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