include thermal shift by differential scanning fluorimetry, fluorescence intensity, microscale thermophoresis, and surface plasmon
resonance.
It is undoubtedly the case that certain techniques are better
suited to some experimental systems than others for a variety of
physical reasons, and that some experimental systems afford a better
chance to showcase the full information content of particular techniques. However, using a single experimental system for as many
techniques as possible illustrates and encourages confirmation of
results by orthogonal assays, and promotes discussion of the
strengths and weaknesses of each type of measurement.
The body of data arising from many repeats of these experiments also provides a basis for discussion of techniques for analysis
of replicate datasets, and for calculation of meaningful confidence
intervals for K d . Not only does this better inform the comparison of
K d between different techniques, but it also illustrates the level of
agreement that can be expected for multi-technique analysis of a
well-behaved and well-characterized system, when measured in a
single location with a single set of instruments. The observed
degree of agreement or variation gives a more realistic framework
within which to evaluate the significance of variation observed for
other projects, either between techniques, or by comparison with
literature.
1.2 A Brief
Introduction to HEWL
and NAG3
Lysozymes are enzymes that cleave the glycosidic bond between
the C-1 of N-acetyl muramic acid and the C-4 of N-acetyl glucosamine in bacterial cell wall peptidoglycan [1]. The apo structure of
HEWL was the first enzyme structure to be solved by X-ray crystallography [2], and the structure of the NAG3-bound form was later
solved by the same group [3]. NAG3 binds in the first three (A–C)
of six subsites in the substrate-binding cleft in wild-type
(WT) HEWL. NAG3 makes many interactions with the protein
including an extensive hydrogen bond network, and hydrophobic
and hydrogen-bonding interactions with two tryptophans (W62,
W63). NAG3 binding causes small changes in the conformation
and dynamics of the protein, particularly in the binding site [4, 5],
and changes in water structure in and around the binding site
[3]. Binding of NAG3 does not result in catalysis because the active
site lies between subsites C and D, so NAG3 acts as a competitive
inhibitor. The association of HEWL and NAG3 has been characterized by calorimetry and absorbance spectroscopy [6–8], giving
estimates for K d in the range 5–10 μM, with experimental conditions varying in the range pH 4.7–5.3 and ionic strength
68–100 mM.
48
Xiaochun Li-Blatter et al.
resonance.
It is undoubtedly the case that certain techniques are better
suited to some experimental systems than others for a variety of
physical reasons, and that some experimental systems afford a better
chance to showcase the full information content of particular techniques. However, using a single experimental system for as many
techniques as possible illustrates and encourages confirmation of
results by orthogonal assays, and promotes discussion of the
strengths and weaknesses of each type of measurement.
The body of data arising from many repeats of these experiments also provides a basis for discussion of techniques for analysis
of replicate datasets, and for calculation of meaningful confidence
intervals for K d . Not only does this better inform the comparison of
K d between different techniques, but it also illustrates the level of
agreement that can be expected for multi-technique analysis of a
well-behaved and well-characterized system, when measured in a
single location with a single set of instruments. The observed
degree of agreement or variation gives a more realistic framework
within which to evaluate the significance of variation observed for
other projects, either between techniques, or by comparison with
literature.
1.2 A Brief
Introduction to HEWL
and NAG3
Lysozymes are enzymes that cleave the glycosidic bond between
the C-1 of N-acetyl muramic acid and the C-4 of N-acetyl glucosamine in bacterial cell wall peptidoglycan [1]. The apo structure of
HEWL was the first enzyme structure to be solved by X-ray crystallography [2], and the structure of the NAG3-bound form was later
solved by the same group [3]. NAG3 binds in the first three (A–C)
of six subsites in the substrate-binding cleft in wild-type
(WT) HEWL. NAG3 makes many interactions with the protein
including an extensive hydrogen bond network, and hydrophobic
and hydrogen-bonding interactions with two tryptophans (W62,
W63). NAG3 binding causes small changes in the conformation
and dynamics of the protein, particularly in the binding site [4, 5],
and changes in water structure in and around the binding site
[3]. Binding of NAG3 does not result in catalysis because the active
site lies between subsites C and D, so NAG3 acts as a competitive
inhibitor. The association of HEWL and NAG3 has been characterized by calorimetry and absorbance spectroscopy [6–8], giving
estimates for K d in the range 5–10 μM, with experimental conditions varying in the range pH 4.7–5.3 and ionic strength
68–100 mM.
48
Xiaochun Li-Blatter et al.