Biophys J, May 1998, p. 2141-2141, Vol. 74, No. 5
NEW AND NOTABLE
Expanding Time Scales Usher in a New Era for Kinetic Studies
Joseph M.
Beechem
Department of Molecular Physiology and Biophysics, Vanderbilt
University, Nashville, Tennessee 37232 USA
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INTRODUCTION |
In 1985 an article was published in
Review of Scientific Instrumentation (vol. 56, 283-290),
titled "Mixing liquids in microseconds," from the laboratory of
Thomas M. Jovin (Max-Planck-Institut für Biophysikalische Chemie,
Göttingen, Germany), authored by Peter Regenfuss, Robert M. Clegg, Mack J. Fulwyler, Francisco J. Barrantes, and Thomas M. Jovin.
This pioneering work described the development of a continuous-flow
microsecond mixing device. This article was clearly published before
its time, and the design languished essentially unused in the
scientific community for the next 9 years or so. I think that there are
quite a few reasons for this, and I will give you one perspective.
The technique of using stopped-flow mixing to examine fast
reactions in the millisecond time scale had reached prominence in the
1960s and 1970s, and by the time the work by Regenfuss et al. (1985)
appeared, emphasis in research had radically shifted from "classical
biophysics" (e.g., kinetics, reaction dynamics, thermodynamics) to
"modern biology" (e.g., protein structure and molecular biology).
This shift in emphasis may have "diluted" the impact of the
development of this important new kinetic tool. The research
"pendulum," however, is beginning to swing back, with renewed
interest being placed on obtaining a more detailed understanding of how
protein structures "actually work" and are assembled (i.e.,
folded). To answer mechanistic questions of this type, kinetic studies
are required, and renewed interest in rapid kinetic methods is once
again surfacing. Pioneering microsecond mixing studies of protein
folding from the laboratories of Denis L. Rousseau (Takahashi et al.,
1995, 1997) and James Hofrichter (Chan et al., 1997) have recently
appeared that utilize a T-mixer version of the original Regenfuss
(1985) design.
In this issue of Biophysical Journal, the
laboratory of Heinreich Roder have an article titled "A
continuous-flow capillary mixing method to monitor reactions on the
microsecond time scale," authored by M. C. Shastry, Stanley D. Luck, and Heinrich Roder. This article provides a detailed description
of the assembly and operation of a modern microsecond mixing,
continuous-flow instrument, and is an adaptation of the original
Regenfuss (1985) design. Using a combination of both continuous-flow
microsecond mixing and conventional stopped-flow mixing, this work
reveals how continuous kinetic data from 50 µs to >10 s after the
initiation of a chemical reaction (in this case, the refolding of
cytochrome c during a chemical jump from pH 2 to pH 4.5) can
be obtained. Kinetic studies of this type should radically enhance our
understanding of the mechanism of protein folding, as well as a wide
variety of other important biological reactions. Although
equilibrium-based methods used to study protein folding are certainly
very useful, it should be remembered that Josiah Willard Gibbs
(1839-1903) formulated the thermodynamic functions to be state
functions, independent of path. Although the concept of a folding
"pathway" has evolved into a more realistic concept of "energy
landscapes" and "folding funnels" (see, e.g., Dill and Chan,
1997) to resolve these complex processes, detailed kinetic
studies over as wide a range of time scales as possible are absolutely
crucial.
Do fast kinetic studies of this type make all of the slower
millisecond mixing experiments obsolete? Certainly not
for processes such as protein folding, it is clear that important kinetic events are
occurring over all of the time scales that have been examined: from
nanoseconds (using the newly developed laser-based T-jump methods), to
microseconds (microsecond mixing; see Shastry et al., this
issue of Biophysical Journal), to milliseconds (conventional stopped-flow mixing), to seconds. One must not loose sight of the fact
that recovery of enzyme activity almost invariably occurs only in the
slowest phase of a refolding experiment. With the availability of
kinetic experiments that can now span almost 10 log units in time, the
real challenge for the future is not so much how to achieve the time
resolution. Instead, what is desperately needed is advanced
spectroscopic detection methodologies and experimental designs that can
be performed in a kinetic mode and yield structural information concerning folding intermediates (e.g., Lillo et al., 1997). Especially lacking is good experimental approaches to
quantifying heterogeneous folding ensembles in terms of something other
than abstract states on a complex energy landscape. Roder's (and
related technologies from other laboratories) microsecond mixing device will greatly help bridge the gap between reaction rate theory (ps/ns)
and biological structure/function (
Å per ps-µs-ms-s).
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REFERENCES |
-
Chan, C.-K.,
Y. Hu,
S. Takahashi,
D. L. Rousseau,
W. A. Eaton, and J. Hofrichter.
1997.
Submillisecond protein folding kinetics studied by ultrarapid mixing.
Proc. Natl. Acad. Sci. USA.
94:1779-1784[Abstract/Full Text].
-
Dill, K. A., and H. S. Chan.
1997.
From Levinthal to pathways to funnels.
Nature Struct. Biol.
4:10-19[Medline].
-
Lillo, M. P.,
B. K. Szpikowska,
M. T. Mas,
J. D. Sutin, and J. M. Beechem.
1997.
Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.
Biochemistry.
36:11273-11281[Medline].
-
Regenfuss, P.,
R. M. Clegg,
M. J. Fulwyler,
F. J. Barrantes, and T. M. Jovin.
1985.
Mixing liquids in microseconds.
Rev. Sci. Instrum.
56:283-290.
-
Takahashi, S.,
Y. C. Ching,
J. Wang, and D. L. Rousseau.
1995.
Microsecond generation of oxygen-bound cytochrome c oxidase by rapid solution mixing.
J. Biol. Chem.
270:8405-8407[Abstract/Full Text].
-
Takahashi, S.,
S.-R. Yeh,
T. K. Das,
C.-K. Chan,
D. S. Gottfried, and D. L. Rousseau.
1997.
Folding of cytochrome c initiated by submillisecond mixing.
Nature Struct. Biol.
4:44-50[Medline].
Biophys J, May 1998, p. 2141-2141, Vol. 74, No. 5
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Introduction
References
