Chemistry Reference
In-Depth Information
Unfortunately, once equilibrium is reached, no more dynamical information is
available.
Single-molecule spectroscopy provides a newway to understand protein dynamics:
isolate the protein and observe that one molecule for a long period of time. This is a
revolutionary concept; its simplicity is one of its strengths, allowing its use in many
different
fields. By watching proteins in action one at a time, we are able to obtain the
previously hidden dynamical information necessary to understand the mechanisms
and limitations of proteinmachines. The goal of our description of proteinmachines
is to move beyond cartoon depiction of protein action. Rather than simply determin-
ing what the moving parts are, we want to know how much friction there is between
the moving parts, how much power is supplied by a chemical reaction, and how the
binding of a another protein affects the action of the protein
fluctuations. We want to
know the order and timing of events.
There are threemajor bene
ts of performingmeasurements at the singlemolecule
level: the abilities to (i) determine distributions of subpoupulations, (ii) measure
long time scale dynamics that are unsynchronized with initial conditions, and
(iii) measure the relative timing of coordinated events, especially those unsynchro-
nized with initial conditions.
9.1.1.1 Distributions of Subpoupulations
Biological systems are in general not homogeneous. An ensemble of proteins may be
in different states. These states may differ in structure, may differ in binding to other
proteins or nucleic acids, or in the progression of a reaction. Single-molecule
methods provide new information on the distributions of these states. Based on
signals from single proteins, molecules can be sorted into the various states,
permitting further study of the properties of these states in isolation.
9.1.1.2 Dynamics of Unsynchronized Trajectories
Biological processes are not random, but thermal
fluctuations do add a random
element to the timing. Random
fluctuations always occur, but there is a non-random
sequence to events; see for example studies monitoring the motion of molecular
motors such as DNA polymerase [6] and myosin [7]. Due to this, synchronization at
the ensemble level is lost very quickly. With single-molecule spectroscopy, it is
possible to answer questions about dynamics of multiple, successive events even in
the presence of randomizing factors.
9.1.1.3 Order of Events/States
Questions related to the order of events and motion on the energy landscape in
between molecular states, are also related to unsynchronized dynamics. Many
biological processes are known to involve binding of two different partners. There
are very few ways to determine the order of binding events. For instance, does A bind
B before or after binding C, or do they all bind at the same time? Also, many enzymes
undergo an ordered series of conformational changes during their catalytic cycle.
Single-molecule spectroscopy allows the unraveling of these binding and/or confor-
mational sequences.
