314
characterization of large protein systems with molecular weights of hundreds of
kDa. This has provided unique insights into the binding, dynamic, and allosteric
properties of enzymes (Huang and Kalodimos 2017).
The useful approach to study enzyme structure by protein NMR is the observation of the resonances from histidine. The C-2 and C-5 proton resonances are downfield from the aromatic protons (Markley 1975). The classical use of these properties
was with the small enzyme (Mr = 23,500) RNAase (Meadows and Jardetzky 1986)
and the large enzyme (Mr = 237,000) pyruvate kinase (Meshitsuka et al. 1981). The
C-2 proton resonance is especially sensitive to the ionization state of the imidazole
nitrogens, thus the pKa for each individual histidine within the native enzyme can
be obtained from titration studies. The binding of a ligand or metal ion to a specific
histidine or histidines could result in a change in the magnetic environment (chemical shift) of the resonance and an alteration in the pKa. This application of NMR has
been useful in some limited number of enzymes. Enzymes enriched with
13
C and
15
N have been used to increase the range of chemical shifts of these nuclei in order
to enhance spectral dispersion and increases the possibility of resolving more resonances. The detailed structural and dynamic studies of larger proteins have been
done with
13
C and
15
N isotope labels through NMR and nuclear Overhauser effect
(Redfield et al. 1989). This type of studies is routine for determining the structure of
enzymes and their dynamics using multidimensional NMR (Kevin et al. 1998;
Bachovchin 2001). An alternative approach is use of a reporter group such as
19
F on
the enzyme or on the substrate to obtain information regarding enzyme structure
and the effects of ligand binding on the enzyme (Geric 1981; Danielson and Falke
1996).
19
F nucleus is 83% as sensitive as
1
H, and has a large range of chemical shifts
in addition there are no back ground resonances of
19
F to cause interference. The
19
F
reporter groups can be incorporated by different methods. A fluorinated amino acid
i.e. fluorotyrosine, fluor-oalanine can be added to growth medium and incorporated
into the protein (Sykes and Weiner 1980). The amino acids i.e. tyrosines, alanines
containing the
19
F are labeled and will exhibit a resonance. The hetero dimer of
tubulin, the principal protein of microtubules, fluoro tyrosine can be incorporated to
α-subunit on the C-terminal amino acid through the reaction catalyzed by tubulin–
tyrosine-ligase (Monasterio et al. 1995). An alternative approach is to covalently
label the enzyme at a specific residue with a fluorine-containing reagent like trifluoroacetic anhydride, trifluoroacetyliodide, or 3-bromo-1, 1, 1-trifluoro-propanone.
The chemical shift and/or the line width (1/T2) of the
19
F label, a “reporter” for a
change in the enzyme structure, must reflect ligand binding and/or catalysis. In case
19
F resonance is sensitive to conformational changes in the enzyme then site- specific
modification of groups at the active site will be reflected by changes in the
19
F resonance. The method of using reporter groups can be also be elucidated by using other
labels like
2
H or
13
C labels. However, most other labels are less sensitive than fluorine. A potential strength of using these labels is the incorporation of
2
H for
1
H or
13
C for
12
C into the protein will have a very minor, if any, effect on the protein itself.
Use of reporter groups yield information regarding the environment of the group.
But not the specific structural features of the enzyme, comparative structural
changes can be studied by photo-chemically induced nuclear polarization (photo
S. A. Rather et al.
characterization of large protein systems with molecular weights of hundreds of
kDa. This has provided unique insights into the binding, dynamic, and allosteric
properties of enzymes (Huang and Kalodimos 2017).
The useful approach to study enzyme structure by protein NMR is the observation of the resonances from histidine. The C-2 and C-5 proton resonances are downfield from the aromatic protons (Markley 1975). The classical use of these properties
was with the small enzyme (Mr = 23,500) RNAase (Meadows and Jardetzky 1986)
and the large enzyme (Mr = 237,000) pyruvate kinase (Meshitsuka et al. 1981). The
C-2 proton resonance is especially sensitive to the ionization state of the imidazole
nitrogens, thus the pKa for each individual histidine within the native enzyme can
be obtained from titration studies. The binding of a ligand or metal ion to a specific
histidine or histidines could result in a change in the magnetic environment (chemical shift) of the resonance and an alteration in the pKa. This application of NMR has
been useful in some limited number of enzymes. Enzymes enriched with
13
C and
15
N have been used to increase the range of chemical shifts of these nuclei in order
to enhance spectral dispersion and increases the possibility of resolving more resonances. The detailed structural and dynamic studies of larger proteins have been
done with
13
C and
15
N isotope labels through NMR and nuclear Overhauser effect
(Redfield et al. 1989). This type of studies is routine for determining the structure of
enzymes and their dynamics using multidimensional NMR (Kevin et al. 1998;
Bachovchin 2001). An alternative approach is use of a reporter group such as
19
F on
the enzyme or on the substrate to obtain information regarding enzyme structure
and the effects of ligand binding on the enzyme (Geric 1981; Danielson and Falke
1996).
19
F nucleus is 83% as sensitive as
1
H, and has a large range of chemical shifts
in addition there are no back ground resonances of
19
F to cause interference. The
19
F
reporter groups can be incorporated by different methods. A fluorinated amino acid
i.e. fluorotyrosine, fluor-oalanine can be added to growth medium and incorporated
into the protein (Sykes and Weiner 1980). The amino acids i.e. tyrosines, alanines
containing the
19
F are labeled and will exhibit a resonance. The hetero dimer of
tubulin, the principal protein of microtubules, fluoro tyrosine can be incorporated to
α-subunit on the C-terminal amino acid through the reaction catalyzed by tubulin–
tyrosine-ligase (Monasterio et al. 1995). An alternative approach is to covalently
label the enzyme at a specific residue with a fluorine-containing reagent like trifluoroacetic anhydride, trifluoroacetyliodide, or 3-bromo-1, 1, 1-trifluoro-propanone.
The chemical shift and/or the line width (1/T2) of the
19
F label, a “reporter” for a
change in the enzyme structure, must reflect ligand binding and/or catalysis. In case
19
F resonance is sensitive to conformational changes in the enzyme then site- specific
modification of groups at the active site will be reflected by changes in the
19
F resonance. The method of using reporter groups can be also be elucidated by using other
labels like
2
H or
13
C labels. However, most other labels are less sensitive than fluorine. A potential strength of using these labels is the incorporation of
2
H for
1
H or
13
C for
12
C into the protein will have a very minor, if any, effect on the protein itself.
Use of reporter groups yield information regarding the environment of the group.
But not the specific structural features of the enzyme, comparative structural
changes can be studied by photo-chemically induced nuclear polarization (photo
S. A. Rather et al.