Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 129

transition states involved in protein complex formation/dissociation and
protein/polypeptide reversible unfolding by both steady-state and rapid
kinetics, and anisotropy analysis. 65 Investigation of protein/protein associa-
tion by mixing the two purified partners in solution and following their as-
sociation in a time-dependent manner is limited to only few examples. 37
Usually, solvent (acetonitrile, dimethyl formamide, isopropanol, etc.) or
chaotropic agents (guandinium chloride, urea, etc.) are used to promote
complex dissociation and protein unfolding. Fluorescence changes can be
transformed to yield the relative fraction of unfolded/monomeric protein
in order to determine the thermodynamic parameters of protein complex
stability. 66 In most cases, fluorescence follows a sigmoidal transition

according to a one-step/two-state model:

D 2

$ 2U = M t ½ 4 : 4

The process can be described by the following equations in which the
folded dimer (D) is at equilibrium with the unfolded monomer (U/ M t ).
The total concentration of monomers ( M t ) at any concentration of solvent
can be defined in terms of the fraction of monomeric protein ( M m ) and K d
can be expressed in terms of measurable values M t and M m .

2

K d ¼ 2 M t MðÞ = ð 1 M m Þ ½ 4 : 5

2

f u 2

K d ¼ ½ U = ½ N 2 ¼ 2 P t = ð 1 f u Þ ½ 4 : 6

The free energy of unfolding/dissociation for a two-statemodel is defined
as a linear function of the concentration of the unfolding/dissociating agent.

DG d ¼DG H 2 O

þ m solvent ½ ¼ RT ln K d ½ 4 : 7

where m corresponds to the slope of the plot of DG d versus [solvent]. [Solvent]
is the concentration of solvent, and R and T are the gas constant and absolute
temperature, respectively. DG d was calculated via K d at the corresponding
concentrations of solvent used. P t corresponds to the total protein concentra-
tion and f u is the fraction of unfolded/monomeric protein. DG H 2 O is the ex-
trapolated free energy of unfolding in the absence of any unfolding agent.
Steady-state fluorescencemeasurements have been combinedwith lifetime
anisotropy to sense protein complex dissociation, 67 as described for the small
protein Dim2, a regulatory component of the splicesome machinery. Analysis
of Dim2 dissociation using guadiniumchloride by steady fluorescence and life-
time anisotropy ( Fig. 4.11 ) has revealed that the small Dim2 protein exists in
two states, monomer and dimer, with a dissociation constant in the nanomolar