169
property of an asymmetric molecular structure wherein the chromofore and the chiral center are covalently fixed and coupled, but rather a consequence of complex
intramolecular transition which favors specific chiral rotamers with strong Cd. A
similar situation may take place when an achiral chromophore binds to protein and
the docking interactions enforce a chiral and therefore optically-active conformation in the the ligand. this induced circular dichroism (ICd, also termed extrinsic
Cotton effect) is usually observed within near-uv or even visible range of light i.e.
at the ligands specific electronic transition (table 6.1). Certainly, induced Cd can
be also triggered under more subtle circumstances—for instance when upon the
binding event the ligand remains rigid and achiral in terms of its covalent structure
but enters chiral fields of the protein, etc. [71]. Examples of applications of ICd
spectroscopy for detection and characterization of protein-ligand interactions in
solution include binding of bilirubin [72] and retinoic acid [73] to β-lactoglobulin.
Another example is ICd of thioflavin t (tht). tht is an important ligand for amyloid fibrils—linear aggregates of misfolded protein molecules which form in vivo
in the course of certain neurodegenerative maladies, such as Alzheimer disease. the
dye has become the most widely used amyloid-specific fluorescent stain, which
according to the prevailing model of amyloid-tht interactions binds specifically to
multistranded β-sheets, such as those constituting amyloid fibrils. In solution, ThT
rotates freely not only as a whole, but also internally around its single C-C bond
linking benzothiazole and dimethylaminobenzene rings. In consequence, timeaveraged conformation of a tht molecule has effectively the Cs symmetry and is
therefore achiral. We have shown earlier that a free tht molecule has two mirrorimage equilibrium structures at the dihedral angle of ± 37°. Stabilization of one of
them by of a chiral environment induces optical activity in the dye [74], as all tht
conformers are chiral except for those perfectly planar, or twisted at the right angle.
this has triggered the interest in using ICd of amyloid-bound tht as a spectroscopic probe of chiral bias of amyloid surface moieties [75–78]. A very interesting
property of tht emerged when the same ICd methodology was used to study interactions of the stain with α-helical poly-L-glutamic acid—an amyloidogenic peptide in a non-fibrillar conformation, which according to the widespread notion was
not supposed to bind the amyloid-specific dye. the data shown in Fig. 6.4 shows,
however, that tht does bind to this non-amyloid conformation, as is revealed by
ICd spectra. It should be stressed that the binding could not be observed through
the fluorescence emission typically employed to detect tht-protein binding, as the
interactions with α-helical poly-L-glutamic acid do not induce enhancement of the
quantum yield of tht fluorescence.
In this brief subsection we have mentioned a number of applications of electronic Cd in respect to the spectral wavelength range employed. Cd brings information
not only on the main chain structure, but also on more subtle effects of packing
intrinsic aromatic chromophores of protein (ruled by the presence of tertiary structure) and extrinsic chromophores binding through non-covalent interactions. We
will show that Cd studies on dNA could be categorized in a similar order.
6 Electronic Circular dichroism Spectroscopy in Structural Analysis …
property of an asymmetric molecular structure wherein the chromofore and the chiral center are covalently fixed and coupled, but rather a consequence of complex
intramolecular transition which favors specific chiral rotamers with strong Cd. A
similar situation may take place when an achiral chromophore binds to protein and
the docking interactions enforce a chiral and therefore optically-active conformation in the the ligand. this induced circular dichroism (ICd, also termed extrinsic
Cotton effect) is usually observed within near-uv or even visible range of light i.e.
at the ligands specific electronic transition (table 6.1). Certainly, induced Cd can
be also triggered under more subtle circumstances—for instance when upon the
binding event the ligand remains rigid and achiral in terms of its covalent structure
but enters chiral fields of the protein, etc. [71]. Examples of applications of ICd
spectroscopy for detection and characterization of protein-ligand interactions in
solution include binding of bilirubin [72] and retinoic acid [73] to β-lactoglobulin.
Another example is ICd of thioflavin t (tht). tht is an important ligand for amyloid fibrils—linear aggregates of misfolded protein molecules which form in vivo
in the course of certain neurodegenerative maladies, such as Alzheimer disease. the
dye has become the most widely used amyloid-specific fluorescent stain, which
according to the prevailing model of amyloid-tht interactions binds specifically to
multistranded β-sheets, such as those constituting amyloid fibrils. In solution, ThT
rotates freely not only as a whole, but also internally around its single C-C bond
linking benzothiazole and dimethylaminobenzene rings. In consequence, timeaveraged conformation of a tht molecule has effectively the Cs symmetry and is
therefore achiral. We have shown earlier that a free tht molecule has two mirrorimage equilibrium structures at the dihedral angle of ± 37°. Stabilization of one of
them by of a chiral environment induces optical activity in the dye [74], as all tht
conformers are chiral except for those perfectly planar, or twisted at the right angle.
this has triggered the interest in using ICd of amyloid-bound tht as a spectroscopic probe of chiral bias of amyloid surface moieties [75–78]. A very interesting
property of tht emerged when the same ICd methodology was used to study interactions of the stain with α-helical poly-L-glutamic acid—an amyloidogenic peptide in a non-fibrillar conformation, which according to the widespread notion was
not supposed to bind the amyloid-specific dye. the data shown in Fig. 6.4 shows,
however, that tht does bind to this non-amyloid conformation, as is revealed by
ICd spectra. It should be stressed that the binding could not be observed through
the fluorescence emission typically employed to detect tht-protein binding, as the
interactions with α-helical poly-L-glutamic acid do not induce enhancement of the
quantum yield of tht fluorescence.
In this brief subsection we have mentioned a number of applications of electronic Cd in respect to the spectral wavelength range employed. Cd brings information
not only on the main chain structure, but also on more subtle effects of packing
intrinsic aromatic chromophores of protein (ruled by the presence of tertiary structure) and extrinsic chromophores binding through non-covalent interactions. We
will show that Cd studies on dNA could be categorized in a similar order.
6 Electronic Circular dichroism Spectroscopy in Structural Analysis …