Disruption of Protein-Protein Interfaces by Stefano Mangani

Author:Stefano Mangani
Language: eng
Format: epub
Publisher: Springer Berlin Heidelberg, Berlin, Heidelberg

In the presence of binding-induced conformational rearrangements, the chemical shift perturbation may extend to residues that are not at the interface, and the chemical shift perturbation fails as a mapping tool, although it still represents an excellent indicator of allosteric processes. Such a situation is associated with slow k on.

Finally, under fast exchange conditions, K d values and stoichiometry can be estimated from the titration of the 15N-labeled observed protein with its unlabeled binding partner. Resonances of the nuclei at the interface shift in a continuous fashion in the HSQC spectra during the titration and fitting the chemical shift perturbation as a function of the concentration of the partner provide affinity constants (Fig. 4.3). If chemical shift changes in the HSQC maps are linear and occur at the same rate for the affected residues, a single binding event is occurring; if changes for different residues occur at different rates and/or trajectories deviate from linearity, more than one binding event/site is implicated. Under the slow exchange regime, during a titration, the set of resonances associated with the free state will decrease in intensity, while that of the bound state will increase in intensity. For residues that are not affected by the interaction, the two sets will coincide. Disappearance of a resonance of the free state from its original position indicates that the corresponding residue is affected by the interaction. An evaluation of the extent of the chemical shift change upon binding will require an independent assignment procedure for the bound state. The affinity constant can be quantitated by fitting the intensity of the disappearing or appearing peaks as a function of the concentration of the added titrant. Intermediate exchange regime is accompanied by resonance broadening, sometimes beyond detection. While observation of this phenomenon provides a proof of the binding event, frequencies of the affected resonances become poorly defined and a detailed spectral analysis may become unfeasible.

Fig. 4.3A plot of the weighted average chemical shift changes of the backbone amide resonances for selected HSQC peaks (L90, G94, G196) in 15N-enriched Bcl-Xl as a function of the concentration of human cytochrome c. Fitting of these data provides the K d value

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