Conformational changes, monitoring techniques

The pKa at high pH is caused by a substrate-independent conformational change in the enzyme, a change that may be monitored directly by physical techniques such as optical rotation and fluorescence yield. Kinetic measurements give the same pKa as that found by these methods. 

Abstract. A recently developed new method to monitor reaction kinetics of intermolecular interaction is reviewed. This method is based on the measurement of the time-dependent diffusion coefficient using the pulsed-laser-induced transient grating technique. Using this method, conformation change, transient association, and transient dissociation on reactions are successfully detected. The principle and some applications to studies on changes in the intermolecular interactions of photosensor proteins (e.g., photoactive yellow protein, phototropins, AppA) in the time domain are described. In particular, unique features of this time-dependent diffusion coefficient method are discussed. 

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

Of the other techniques to follow conformational changes, dynamic light scattering gives the diffusion coefficient, which will decrease with the size of the structure, due to an increase in the frictional coefficient. UV difference spectroscopy can be used to monitor... 

This study addresses the question of how bulk polymer chemistry and surface energy affect the amount and the conformation of FN adsorbed to a series of polyurethaneureas. The technique of Fourier transform infrared spectroscopy (FTIR) coupled with attenuated total reflectance (ATR) optics was used to continuously and non-invasively measure the kinetics of FN adsorption, as well as to monitor conformational changes occuring during adsorption. 

SSCP is a popular technique for the screening of mutations or polymorphisms in short segments of DNA. Mutations are detected by monitoring of mobility shifts of individual DNA strands caused by their conformation changes. The main advantage of the SSCP technique is the ability to detect mutations (substitutions, small deletions, and insertions) at a variety of positions in DNA fragments with good... 

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