Proteins conformational changes, monitoring

In spite of the lack of a detailed correlation between the photochemical events as monitored spectroscopically and the electrical events in the retina, it is generally believed that the transmitter release and the subsequent LRP are initiated by protein conformational change occurring in the MI478 + MIl3gQ transition (337,338). The evidence in this respect comes from the observation that the preceding thermal processes, BAT543 - ... 

The simplicity of performing this protein secondary structure analysis makes CD spectroscopy useM in monitoring protein conformational changes between wild-type and mutant proteins, as well as in identifying changes in the protein secondary structure upon addition of a bound cofactor such as a metal ion. Several recent examples of applying CD spectroscopy for these purposes are now described. 

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. 

Jang, D. J. Elsayed, M. A. Tryptophan fluorescence quenching as a monitor for the protein conformation changes occurring during the photocycle of hacteriorhodopsin under different perturbations. Proc. Nat. Acad. Set U.S.A., 1989, 86(15), 5815-5819. 

Despite its weakness, the anisotropy of the g tensor of iron-sulfur centers can be used to determine the orientation of these centers or that of the accommodating polypeptide in relation to a more complex system such as a membrane-bound complex. For this purpose, the EPR study has to be carried out on either partially or fully oriented systems (oriented membranes or monocrystals, respectively). Lastly, the sensitivity of the EPR spectra of iron-sulfur centers to structural changes can be utilized to monitor the conformational changes induced in the protein by different factors, such as the pH and the ionic strength of the solvent or the binding of substrates and inhibitors. We return to the latter point in Section IV. 

Calleja, V., Ameer-Beg, S. M., Yojnovic, B., Woscholski, R., Downward, J. and Larijani, B. (2003). Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem. J. 372, 33 40. 

The hydrolysis of mant-GTP bound to Ras can be monitored by a slight decrease in fluorescence. Binding experiments of N-Ras with the non-hydro-lyzable GTP-analogue mant-GppNHp showed a biphasic increase in fluorescence. The slow phase had the same amplitude as the decrease observed for the hydrolysis of Ras mant-GTP which led to the hypothesis that a conformational change in the Ras protein proceeds GTP-hydrolysis [170] and represents the rate limiting step ... 

The application of heterobifunctional cross-linkers allows macromolecular PAL to probe protein-protein interactions, including subunit interactions and location, monitoring the conformational changes induced by signal transmission. 

Fig. 4. Time-induced conformational change of spider silk protein (spidroin) in solution. Solutions of silk proteins at 1% w/v in distilled water were monitored using circular dichroism. The graph shows a change in secondary structure with time. The silk proteins underwent a kinetically driven transition from a partially unfolded structure to a -sheet-rich structure (from Dicko et al., 2004c). ( ) after 0 days, (O) after 1 day, and (A) after 2 days. The conformational change appeared faster at 20°C compared to 5°C, suggesting a hydrophobically driven mechanism. (Copyright 2004 American Chemical Society.)...

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