Chemistry Reference
In-Depth Information
We focus this chapter on measuring protein dynamics using uorescence. For
some (mechanical-based) questions, single-molecule force experiments (laser and
magnetic tweezers, AFM) are more appropriate; however, fluorescence-based single-
molecule experiments have several unique bene ts. Fluorescence is minimally
invasive, more generally applicable, and many of the techniques can be extended to
work inside living cells, and possibly inside living tissues and even small organisms.
We plan to provide a researcher outside the single-molecule field with an
understanding of what questions regarding protein dynamics and interactions are
ideally answered using single-molecule
fluorescence methods. In order to illustrate
the use of single-molecule spectroscopy for monitoring protein dynamics and
interactions, we have chosen two example areas: protein-folding and DNA-proces-
sing enzymes. We plan to help the researcher understand the bewildering array of
single-molecule methodologies, what motivates their development and how to
choose the methodology best suited to a particular question.
9.1.2
Example Biological Systems
The ability of single-molecule detection to separate signals from different conforma-
tions of a molecule (e.g. folded and unfolded) and to quantify their respective
proportions under conditions of their coexistence can be exploited to study several
problems that would otherwise hardly be addressable at the ensemble level. Rather
than attempt to survey all of the single-molecule literature regarding protein
dynamics and interactions, we have chosen to use a few examples from our research
to illustrate how these experiments are motivated and designed. We hope that, in
describing our dif culties, mistakes, and successes, newcomers to this exciting eld
will have a better knowledge of what awaits them.
Proteins are not like clay that can be molded into any shape; they contain structural
information in their sequence of amino acids, and fold into precise structures. The
overriding question in the protein folding field is how proteins fold based on nothing
more than the sequence. In protein folding, single-molecule fluorescence studies
give access to the structure and conformational changes in the denatured sub-
ensemble, polypeptide chain collapse under a variety of solvent conditions, and
thermodynamic parameters of the unfolding process. The protein folding field has
already bene ted from the abilities of single-molecule spectroscopy to distinguish
between the multiple species present and to monitor the folding and unfolding of
single proteins over extended periods.
We will also illustrate the application of single-molecule spectroscopy to research
into motion and interactions of DNA processing enzymes, particularly RNA poly-
merase (RNAP). In order to understand how DNA processing enzymes start,
perform, regulate, and finish their tasks, it is necessary to elucidate the speci c
dynamic details of ordering and movement. Already, many questions have been
answered or are being answered using single-molecule spectroscopy. For instance,
how long does the RNApolymerase remain attached to a promoter? What is the drag
on the polymerases - how much molecular friction is there between components?
 
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