State of ligation

There are numerous cases of proteins for which structures have been determined in more than one state of ligation. In some cases, the structures undergo little change, except perhaps for specific and localized changes associated with particular functional residues—for example, triose phosphate isomerase. Other cases, such as... 

One can notice that while a shift is observed only for two mutants, the fractional intensities obtained from the lifetime measiu cments are modified in the wild type and in the three mutants (Table 2.7). Emission peak of Tryptophan 4 is not modified with the state of ligation, however fractional intensities ft and increase while f3 is not modified. Emission of tryptophan 170 is located at 338 nm characterizing an emission from a slightly hydrophobic environment. Modification of the state of ligation, Mg instead of Ca, and in the apo form, a red shift to 341 and 350 nm is observed. This means that conformational changes are observed in the vicinity of Trp 170 that is more exposed to the solvent in presence of Mg than in presence of Ca. In the apo form, important structural modifications occur within the protein. 

The >imax of tryptophan 57 in the Ca state is very blue-shifted (317 nm). In presence of Mg " and in the apoform, the maximum shifts to 324 and 330 nm, respectively. Although this red shift indicates a modification in the microenvironment of the Trp residue, the positions of the maximum reveal that Trp 57 is buried in the hydrophobic core of the protein. We observe also that the fractional intensities of the fluorescence lifetimes are not the same with the state of ligation. The mean fluorescence lifetime is drastically modified when the protein goes from the apo to the ligated forms. 

In the wild-type protein, while no shift is observed in the emission peak, the fractional intensities of the fluorescence lifetimes vary with the state of ligation. 

The three Trp residues in NSCP can be used to monitor structural and / or dynamical modifications with the state of ligation of the protein. 

Emission from two rigid species would imply that the fraetional eontributions of eaeh fluoreseenee lifetime should be the same whatever the state of ligation, whieh is not the ease (Table 9.2). 

In this case, the signal monitors the progress of the saturation of a macromolecule and a normal titration (addition of a ligand to a constant macromolecule concentration) is generally performed. Any physico-chemical intensive property of the macromolecule (e.g. fluorescence intensity, fluorescence anisotropy, absorbance, circular dichroism, viscosity, etc.) can be used to monitor the binding, if this property is affected by the state of ligation of the macromolecule. 

Gouaux, J.E., Lipscomb, W.N. Crystal structures of phosphonoacetamide ligated T and phosphono-acetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8 A resolution and neutral pH. Biochemistry 29 389-402, 1990. 

A relationship between the redox state of an iron—sulfur center and the conformation of the host protein was furthermore established in an X-ray crystal study on center P in Azotobacter vinelandii nitroge-nase (270). In this enzyme, the two-electron oxidation of center P was found to be accompanied by a significant displacement of about 1 A of two iron atoms. In both cases, this displacement was associated with an additional ligation provided by a serine residue and the amide nitrogen of a cysteine residue, respectively. Since these two residues are protonable, it has been suggested that this structural change might help to synchronize the transfer of electrons and protons to the Fe-Mo cofactor of the enzyme (270). 

The above procedure readily yields Fig. 9.2. For estimating the excess chemical potential in the protein, again we need to know the ligation state of the metal ion. This is well known for the zinc-finger case. So we followed the above procedure, deciding what clusters to study quantum mechanically, and then composing the free energies as above. 

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