temperature of measurement to try and obtain a k obs value
that is within the accessible range (<400 s
À1 ). If brighter
external fluorescent probes are employed then 50 nM of A
and 500 nM of B may be a good starting point with the
reduction in [B] leading to a slower binding reaction and
easier to measure values.
10. We recommend separate preliminary experiments to confirming the presence of different kinetic phases that may be over
different time regimes. However, when the experimental protocol is optimized and the kinetics of a concentration series is
recorded, it may be useful to acquire data with a logarithmic or
split time base to distribute enough data points for each kinetic
phase within the one experiment. Curve fitting of the kinetic
transient may be biased depending on the nonuniform distribution of data points (linear, split, logarithmic) and residuals
must therefore be carefully analyzed for systematic deviations
with this in mind.
11. In general, certain salts promote “structuring” in proteins
according to the Hofmeister series [33]. Sodium sulfate is
particularly useful for stabilizing weakly structured intermediates that often contain only a collapsed hydrophobic core (for
example, in protein folding). Likewise, trimethylamine Noxide (TMAO) promotes tertiary structure in proteins whereas
2,2,2-trifluoroethanol (TFE) is more specific for stabilizing
hydrogen bonds and thereby stabilizes helical elements. On
the other hand, denaturants such as urea or guanidinium chloride can be used to destabilize protein structure and favor
unfolded states. The additives glycerol and sucrose are sometimes used to increase solution viscosity. This is a common way
to investigate association reactions and determine if they are
diffusion limited since molecular encounters will slow with
increased viscosity. However, their use is complicated because
the increased viscosity will affect the instrument mixing time
(important for fast reactions) and they may also affect the
stability of folded conformations.
12. The concentration of AB is dependent on K d for the interaction and the concentrations of A and B. In general, low concentrations of A and B and a high relative concentration of AB
complex are preferable in a displacement experiment. The
limiting factors are the K d value and the difference in fluorescence between AB and AC. As a useful starting point, if
K d ¼ 100 nM, the concentrations of A and B should be
0.1–1 μM (resulting in 38–73% complex if [A] is equal to
[B]). If K d ¼ 1 μM, then the concentrations of A and B should
be between 1 and 10 μM, etc. With lower concentrations of B,
less C is needed to compete out B, but there will be more
free A, which will give an additional kinetic binding phase not
130
Elin Karlsson and Per Jemth
that is within the accessible range (<400 s
À1 ). If brighter
external fluorescent probes are employed then 50 nM of A
and 500 nM of B may be a good starting point with the
reduction in [B] leading to a slower binding reaction and
easier to measure values.
10. We recommend separate preliminary experiments to confirming the presence of different kinetic phases that may be over
different time regimes. However, when the experimental protocol is optimized and the kinetics of a concentration series is
recorded, it may be useful to acquire data with a logarithmic or
split time base to distribute enough data points for each kinetic
phase within the one experiment. Curve fitting of the kinetic
transient may be biased depending on the nonuniform distribution of data points (linear, split, logarithmic) and residuals
must therefore be carefully analyzed for systematic deviations
with this in mind.
11. In general, certain salts promote “structuring” in proteins
according to the Hofmeister series [33]. Sodium sulfate is
particularly useful for stabilizing weakly structured intermediates that often contain only a collapsed hydrophobic core (for
example, in protein folding). Likewise, trimethylamine Noxide (TMAO) promotes tertiary structure in proteins whereas
2,2,2-trifluoroethanol (TFE) is more specific for stabilizing
hydrogen bonds and thereby stabilizes helical elements. On
the other hand, denaturants such as urea or guanidinium chloride can be used to destabilize protein structure and favor
unfolded states. The additives glycerol and sucrose are sometimes used to increase solution viscosity. This is a common way
to investigate association reactions and determine if they are
diffusion limited since molecular encounters will slow with
increased viscosity. However, their use is complicated because
the increased viscosity will affect the instrument mixing time
(important for fast reactions) and they may also affect the
stability of folded conformations.
12. The concentration of AB is dependent on K d for the interaction and the concentrations of A and B. In general, low concentrations of A and B and a high relative concentration of AB
complex are preferable in a displacement experiment. The
limiting factors are the K d value and the difference in fluorescence between AB and AC. As a useful starting point, if
K d ¼ 100 nM, the concentrations of A and B should be
0.1–1 μM (resulting in 38–73% complex if [A] is equal to
[B]). If K d ¼ 1 μM, then the concentrations of A and B should
be between 1 and 10 μM, etc. With lower concentrations of B,
less C is needed to compete out B, but there will be more
free A, which will give an additional kinetic binding phase not
130
Elin Karlsson and Per Jemth