Cysteine mutagenesis

Brocke, L., Bendahan, A., Grunewald, M., and Kanner, B. I. (2002) Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis. J. Biol. Chem. 277, 3985-3992. 

Simoes, M., Gameau, L., Klein, H., Banderali, U., Hobeila, F., Roux, B., Parent, L., and Sauve, R. 2002. Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel. J. Gen. Physiol. 120 99-116. 

Wo, Z. G., Chohan, K. K., Chen, H., Sutcliffe, M.J., and Oswald, R. E. (1999). Cysteine mutagenesis and homology modeling of the ligand-binding site of a kainate-binding protein./. Biol. Chem. 274, 37210-37218. 

The puriflcation of the y-secretase complex (59) has allowed the first glimpse into its structure. Electron microscopy and single particle analysis reveals that the complex has a globular structure that at low resolution (10-15 A) appears rather amorphous (66). [Another structure, elucidated in a similar manner but of poorer resolution ( 45 A), also has been reported (67).] Nevertheless, two important features can be gleaned. The first is a rather large interior cavity of 20 A diameter that is presumably where the active site resides, a characteristic reminiscent of the proteasome. The second is the presence of two small openings that may be the site of entry for water. Other structural features have been revealed by cysteine mutagenesis with... 

Luchette, P. A., Prosser, R. S. and Sanders, C. R. (2002) Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and F-19 NMR spectroscopy. Journal of the American Chemical Society, 124(8), 1778-1781. 

In addition to photonic switches, photonic proteins were engineered to contain fluorescent moieties that react to changes in a protein s local environment and to conformational changes. In this section, we review several examples of designing photonic probes that goes beyond simple chemical labeling and cysteine mutagenesis. 

Ohmiya, Y., and Tsuji, F. I. (1997). Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity. FEBS Lett. 404 115-117. 

The Fe proteins are homodimers containing a single Fe4S4 cluster. Site-directed mutagenesis experiments showed that the cluster was probably held between the two subunits by ligation to two of the invariant cysteine residues from each subunit (16). This observation was confirmed later by X-ray crystallography (1) of the Fe protein... 

Clusters Fa and Fb of photosystem I from cyanobacteria and chloro-plasts are distinguished by their EPR signatures (26, 27) and their reduction potentials (-520 mV for Fa and -580 mV for Fb Ref. (28). The assignment of cysteines in the primary sequence as ligands to individual clusters has been achieved by site-specific mutagenesis (29, Fig. 3), and structural information with regard to the environment of both clusters has been obtained by NMR (24). 

DNA sequence indicated that AMDase contains four cysteine residues located at 101, 148, 171 and 188 from amino terminal (Eig. 9). At least one of these four is estimated to play an essential role in the decarboxylation. The most effective way to determine which Cys is responsible to enzyme activity will be site-directed mutagenesis. To determine which amino acid should be introduced in place of active Cys, its role was estimated as illustrated in Eig. 13. One possibility is that... 

The basic reactions of thiolsulfonates have been known for sometime (Field et al., 1961, 1964), but more recently, they have been applied to the study of protein interactions by site-directed modification of native cysteines or through modification of cysteines introduced at particular points in proteins by mutagenesis. Such studies have yielded insights into the structure and binding site characteristics of proteins (Kirley, 1989). Pascual et al. (1998) used AEAETS to probe the acetylcholine receptor from the extracellular side of the membrane in order to investigate the molecular accessibility and electrostatic potential within the open and closed channel. 

Zecherle, G.N. (1990) The ribosomal location and conformation of Escherichia coli protein L7/L12 studied by cysteine site directed mutagenesis and crosslinking. Doctoral Dissertation. University of California at Davis. 

LANZ T., TROPF, S., MARNER, F.J, SCHRODER, J., SCHRODER, G., The role of cysteines in polyketide synthases site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways, J. Biol. Chem., 1991, 266, 9971-9976. 

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