Page 149 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013H-614 July 27, 2001 10:29

Protein Folding 189

D. Kinetics Experiments [d], S ∞,[d] , is the signal at equilibrium (infinite time af-
ter initiation of reaction) at the denaturing condition, and
Studying the kinetics of folding or unfolding a protein in-
S i ,[d] is the signal amplitude associated with relaxation
volves the rapid initiation of the reaction by either removal
time constant, τ i . If there is only a single step in the pro-
of the denaturing condition (to initiate folding) or addition
cess (i = 1), the reaction will be a mono-exponential. With
of the denaturing condition (unfolding). The most com-
more steps, the reaction becomes bi-, tri-, .. . exponential,
mon way in which this is done is by a stopped-flow mixing
and fitting the above equation to the data can be difficult.
device, in which a solution of the protein in one solution
Even with the number of terms and the corresponding val-
condition (e.g., neutral buffer with no chemical denatu-
ues of τ i determined, it is still a challenge to relate the τ i
rant) is rapidly mixed with a second solution (e.g., concen-
and the associated amplitudes to a reaction mechanism.
trated chemical denaturant or acid). Temperature or pres-
How this can be done is beyond the scope of this entry
sure can also be used as the perturbing condition. Upward
and we refer readers to References 35 and 36.
temperature jumps can be initiated by high-powered laser
To experimentally apply the above equation, it is neces-
pulses or electrical discharges in the solution. Downward
sary to monitor some signal that responds to the structural
pressure jumps can be initiated by releasing some type
state of the protein, just as is the case in equilibrium stud-
of valve after high pressure has been established. Other
ies. For very rapid folding/unfolding reactions, the moni-
ways to rapidly initiate a protein folding or unfolding re-
toring method must itself be able to respond more rapidly
action include such things as laser flash-induced chemical
(with adequate signal to noise) than the chemical reac-
reactions, which can dissociate heme-carbon monoxide
tion. Fluorescence, absorbance, and circular dichroism
bonds of heme proteins. The remaining discussion will
are fairly rapidly responding optical methods and enjoy
emphasize stopped-flow mixing reactions, since these are
widespread use in combination with stopped-flow mix-
the most widely available approaches.
ers for this purpose. As indicated in Table II, some of the
For small, globular proteins, the kinetics of denaturant-
other methods are not easily adapted for the rapid initiation
induced or acid-induced unfolding reactions are often
and continuous monitoring of a reaction’s progress. Obvi-
found to be described as a mono-exponential process, in-
ously, when a reaction occurs on the time scale of minutes
dicating that there is a single energy barrier between the
to hours, then a hand-mixing experiment can suffice.
native and unfolded species. For larger proteins, partic-
ularly those with multiple domain structures, one often
finds the kinetics to be more complicated. In those cases,
the folding or unfolding reaction may be described by V. CLOSING COMMENTS
more than one exponential decay term. This can be an
The empirical approaches mentioned above have afforded
indication of the existence of multiple, slowly intercon-
great insight into the transition from a linear sequence
verting unfolded states, the existence of multiple energy
of amino acids into a final tertiary structure. Researchers
barriers along the folding/unfolding pathway, the exis-
in the field continue to pioneer new techniques that give
tence of more than one pathway, or the existence of some
insight into the complex inter- and intramolecular interac-
off-path (or dead-end) species. The challenge in kinetics
tions that dictate structure and function of proteins. These
experiments is to first determine the minimum number of
efforts coupled with computational approaches are help-
decay terms needed to describe the reaction and to then
ingtorevealtherulesthatnatureusestocreatethecomplex
determine a reaction mechanism consistent with the kinet-
and unique structure of proteins.
ics data. Often, unique mechanisms cannot be determined
and it is the art of the scientist to establish which mecha-
nism is most reasonable for the system being studied. In
many cases research will focus on determining the num- SEE ALSO THE FOLLOWING ARTICLES
ber of intermediates on the folding pathway and in trying
BIOPOLYMERS • CHEMICAL THERMODYNAMICS • ELEC-
to gain structural information about these intermediates.
TROPHORESIS • NUCLEAR MAGNETIC RESONANCE • PRO-
Whether studying folding or unfolding, the reaction
should be described with the following general empiri- TEIN STRUCTURE • TRANSLATION OF RNA TO PROTEIN
cal relationship: 33,34

S(t) [d] = S ∞,[d] + S i ,[d] · exp(−t /τ i ) (13) BIBLIOGRAPHY
i
Hynes, T. R., and Fox, R. O. (1991). “The crystal structure of staphy-
where S(t) [d] is a time-dependent change in some gen- lococcal nuclease refined at 1.7 A resolution,” Proteins Struct. Funct.
eralized signal at denaturing condition described by Genet. 10, 92–105.