Protein Dynamics and Interactions - Single Molecule Dynamics in Life Science - page 200

two main observables) observed in an experiment, should be carefully analyzed to
exclude genuine photophysical effects that have nothing to do with potential intra- or
intermolecular changes of the macromolecule to which the fluorophore is attached.
Simple controls such as, for instance, studying the excitation power dependence of
the observed effect, analysis of the photophysics of the dye alone or of singly-labeled
macromolecules in the case of FRET studies, are necessary to ascertain the physical
origin of the observable variation (see e.g. [24]).
9.3
Single-molecule Data Acquisition and Analysis Methods
The number of fluorescence-based single-molecule methodologies can be daunting
for newcomers to the field. Nearly every time a new question is approached, a new
methodology is also developed. In order to understand why this is so, an under-
standing of the motivating factors in the development of these methods must be
derived. Depending on the question of interest, the proper method can usually be
found. Or, can be developed as a combination of previous methods (and given a new
name!).
There is only one chance to look at each molecule. In order to maximize the
amount of information obtained from that molecule, experimental schemes of
increasing complexity have been devised. Also, single-molecule measurements
require the same types of controls as ensemble experiments. Many of these controls
must be performed simultaneously with the single-molecule experiments in order to
be meaningful.
Single molecule detection and analysis of protein dynamics and interactions
requires three components: an observable for answering the question of interest,
suf cient signal, and isolation of the signal fromany other signals. In this section, we
will discuss how to satisfy these three requirements, and how to acquire and analyze
the data once the molecules are prepared.
9.3.1
Choosing a Labeling Configuration: What is the Observable?
The choice of observable must be chosen speci cally to answer each new question.
Example labeling con gurations are shown in Figure 9.3. Single-molecule methods
have the largest impact in measuring dynamic changes in the conformation and
interactions of proteins. For observables monitoring protein dynamics and interac-
tions, the diagrams and suggestions suggested in a previous review still apply [25]. In
choosing the observable, the first choice to be made is to determine whether the
conformation of an individual protein will be observed, or whether the con guration
of protein interactions will be observed. Examples of the former include protein
folding measurements and conformational changes induced by protein interactions
such as changes in DNA structure upon binding of CAP [26]. Examples of the latter
include the motion of RNA polymerase on DNA and antibody - antigen interactions.