Cysteine scanning mutagenesis

Site-directed mutagenesis, cysteine-scanning accessibility methods, high field NMR, homology modeling, and continued development of structure-activity relationships will no doubt lead to better and better models of the monoamine transporters. These approaches are having the greatest impact on studies of uptake inhibitors, rather than studies of substrates. 

Loo, T.W. and Clarke, D.M. (1999) Determining the structure and mechanism of the human multidrug resistance P-glycoprotein using cysteine-scanning mutagenesis and thiol-modification techniques. Biochimica et Biophysica Acta, 1461, 315-325. 

Cysteine Scanning Mutagenesis Mapping Binding Sites of Ligand-Gated Ion Channels... 

Cysteine scanning mutagenesis mapping binding sites of ligand-gated ion channels... 

Sullivan D, Chiara DC, Cohen JB. 2002. Mapping the agonist binding site of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis antagonist footprint and secondary structure predictions. Mol Pharmacol 61 ... 

Grunewald M., Menaker D., and Kanner B. I. (2002). Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1. J. Biol. Chem. 277 26074-26080. 

Loo TW, Clarke DM. Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane. J Biol Chem 2000 275(50) 39272-39278. 

Cysteine-scanning mutagenesis, involving more than 100 mutations, has been systematically carried out through Cl—C3, the cytoplasmic terminations of TM1-TM7, H8, and the C-terminal tail. In addition, more than 40 pairs of cysteines have been introduced at the cytoplasmic face. With these mutants as a basis set, three classes of experiments have been carried out, namely SDSL, sulfhydryl reactivity, and disulfide cross-linking kinetics. A global comparison of the results provides a unique view of the solution state, its dynamics, and its correlation with the crystal structure. By solution state is meant, in all cases, rhodopsin solubilized in dodecyl maltoside (DM) micelles. The measured functional properties of rhodopsin, namely transducin activation (Resek et al., 1993) and phosphorylation by the rhodopsin kinase (Thurmond et al., 1997), are conserved in this detergent, and it is presumed to be a reasonable approximation to the bilayer environment. 

C2 constitutes part of the transducin interaction domain (Fig. IB). Figure 10A shows the sequence in C2, TM3, and TM4 (136-155) that has been investigated by cysteine scanning mutagenesis and SDSL (Ridge et at., 1995 Farahbakhsh et al., 1995). Figure 10B (upper panel) shows the fractional solvent accessibilities for residues in the 136-155 sequence... 

As described in the previous sections, cysteine scanning mutagenesis and the associated techniques of SDSL, sulfhydryl reactivity, and disulfide cross-linking rates have provided a rather detailed view of rhodopsin dynamics in solution and conformational changes leading to the activated state. In this section, the structural origins of these functional properties in solution are examined from the point of view of the crystal structure. 

Residues putatively involved in the drug recognition process have therefore been identified via a combination of direct photolabeling, cysteine scanning mutagenesis, and chemical cross-linking (Table 1.1). 

Kurz, L. L., Zuhlke, R. D., Zhang, H. J., andjoho, R. H. (1995). Side-chain accessibilities in the pore of a K- - channel probed by sulfhydryl-specific regents after cysteine-scanning mutagenesis. Biophys. J. 68, 900-905. 

A related fibril model for A/ o was proposed based on scanning proline mutagenesis (Williams et al, 2004) and molecular modeling (Guo et al., 2004). This model proposes that residues 15-21, 24-28, and 31-36 form 3 /-strands, with 2 intervening turns formed by residues 22-23 and 29-30 (Fig. 17G). Residues 17 and 34 are placed in close proximity, as double cysteine mutants at these positions form disulfide bonds on oxidation after fibrillization (Shivaprasad and Wetzel, 2004). Since fibrils with this triangular cross section would not be expected to show an H0-A... 

---