Protein Dynamics and Interactions - Single Molecule Dynamics in Life Science - page 215

protein folding and macromolecular interactions i.e. DNA processing enzymes
(protein-DNA interactions).

9.4.1
Single-molecule Fluorescence Studies of Protein Folding and Conformations

Proteins contain structural information in their sequence and fold spontaneously
into speci c structures. In protein folding studies, folded, unfolded, and partially
folded species may be simultaneously present and rapidly inter-converting, obscur-
ing the properties of individual species. Due to the ability of single-molecule
spectroscopy to sort proteins into different conformational subspecies, single-mole-
cule fluorescence methods are beginning to impact the protein- folding field in a
substantial manner [8]. The properties of the unfolded, and folded, and intermediate
states can be studied individually even when both are simultaneously present. In
immobilized experiments, the timing and dynamics of the folding and unfolding
processes may be observed directly over extended periods.

9.4.1.1 Observables for Protein Folding
In order to apply single-molecule spectroscopy to protein folding, appropriate
observables and labeling schemes must be chosen. One of the main differences
between folded, unfolded, and intermediate states is the size of the overall protein.
FRET, with its ability tomeasure the distance between two points, is a primary tool for
single-molecule measurements of protein folding. Since folding of small proteins is
accomplished by each protein independently, all labels and immobilization attach-
ment pointsmust be on the same protein in order to provide a useful observable. So
far, almost all single-molecule protein folding studies use a scheme where each
protein has one donor and one acceptor attached (Figure 9.3). This has been
adequate for the simple two-state folders studied so far. However, more complex
situations can be imagined. For example, recently developed three-color single
molecule methods may find use in determining the ordering in folding of larger,

more complex proteins: howdoes the folding of domain 1 (measured by FRET from
donor D to acceptor 1) affect the folding of domain 2 (measured by FRET from
donor D to acceptor 2)?
For studies of short-range dynamics involved in protein folding, another possible
observable is electron transfer or fluorescent quenching of a single uorophore.
Changes in quantumef ciency of a uorophore would then indicate contact between
two sites of a protein. Using correlation analysis, even very fast dynamics may be
monitored in this way.

9.4.1.2 Labeling Schemes for Protein Folding
For protein folding studies at the single-molecule level, proteins must be site-
speci cally labeled by donor and acceptor uorophores, and, if immobilized, attached
at a speci c point. Such extensive external modi cation requires careful planning and
extensive work. Several clever strategies have been used to accomplish this complex
labeling.