Chemistry Reference
In-Depth Information
monitor the distance between two proteins or a protein and DNA. Fluorescence
polarization may also be used to observe changes in binding, although expected
changes in anisotropy are usually less dramatic than changes in FRET or ET. One
additional propertymay be used tomonitor changes in interactions: co-localization of
proteins. If a protein is bound to DNA or another protein and each component is
labeled, the signal fromthe various
uorophores are co-localized, andwill either be in
close proximity (if the system is not moving) or will diffuse together (in solution-
based experiments). These signals are extremely important for monitoring the
presence of various components over time.
Once the choice of observable ismade, themore dif
cult task of actually producing
the labeled con
guration is next [27
-
29]. In labeling themolecules, three thingsmust
be borne inmind. First, the speci
city and ef
ciency of the labelingmust be suf
cient
to remove ambiguity in the single-molecule measurements. The most common
method for labeling proteins is a cysteine-based labeling. Labeling of DNA is
generally less dif
cult due to automated synthesis and may often be purchased
ready labeled. Second, does the presence of the
uorophore, or changes made to the
protein or DNA to facilitate labeling affect its function? Third, does the system
actually show the changes or movement expected at the single-molecule level? Even
in cases where the
final question requires immobilization, we
find that solution-
based techniques are often preferable in showing that the observables actually work.
After choosing labeling sites, it is necessary to decide onwhich
uorophores will be
used. This is an important decision as good performance from
fluorophores is
required to obtain useful information from single-molecule studies. In addition to
more traditional dyes such as tetramethylrhodamine (TMR), many excellent
uor-
ophores have become available in recent years. Series of
fluorophores from Atto-tec
and Molecular Probes (Alexa
fluor) are in general excellent choices. In choosing the
fluorophores, it is generally best to start with
fluorophores used successfully
elsewhere. However, issues to consider are extinction coef
cient (how strong the
absorption of light is), quantum ef
ciency (how many excitations lead to
uores-
cence), resistance to photobleaching, sources of background for the laser wave-
lengths chosen, and, when monitoring multiple colors, the extent of overlap of
emission spectra, and the Forster radii of the
uorophores.
9.3.2
Should a Freely-diffusing or Immobilized Format be used?
Next to the labeling con
guration, the most important decision to make in a single-
moleculemeasurement is whether to carry out themeasurements on freely diffusing
molecules in solution or immobilized molecules. The determining factor for which
type of measurement to use is the length of the observation time required to answer
the question at hand.
For example, watching a single protein fold and unfold requires immobilization
since thewaiting times between folding and unfolding events can be in thems
-
s time
scale. However, studying the sub-ms structural
fluctuations of unfolded proteins
does not require immobilization.
