Endogenous Cysteine Residues Research Articles

A Method of Screening for Mutant Proteins Containing Cysteine Residues Using Fluorescein-5-maleimide

A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described. After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS–PAGE gels under ultraviolet light as fluorescent bands. If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence. In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility. This effect permits the detection of cysteine mutations even when endogenous cysteines are present. The described method allows the rapid screening of a large number of transformants.

Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties

We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.

Structure-activity relationship in the gastric cytoprotective effect of several sesquiterpene lactones.

The structural requirements for the gastric cytoprotective activity of several sesquiterpene lactones are reported. A theoretical-experimental study on the potentially active centers is carried out. The biological evaluation of reduced analogues and the simulation of the molecular interactions between these compounds and an endogenous cysteine residue suggest that the presence of a non sterically hindered Michael acceptor seems to be an essential structural requirement for the cytoprotective activity in this family of compounds. This observation suggests that cytoprotection is mediated through a Michael reaction between the sulfhydryl-containing peptides of the mucosa and Michael acceptors present in the molecules under study. This mechanism of action is in addition to and distinct from the one proposed in our previous paper, namely, that these sesquiterpenes stimulate endogenous synthesis of prostaglandins.

Engineered cysteine mutants of myosin light chain 2. Fluorescent analogues for structural studies.

Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine residues at positions 126 and 155; a third sulfhydryl was added by replacing either Pro2, Ser73, or Pro94 with cysteine. By oxidizing the endogenous cysteines to an intramolecular disulfide bond (Katoh, T., and Lowey, S., (1989) J. Cell Biol. 109, 1549), it was expected that the new cysteine could be selectively labeled with a fluorescent probe. This proved more difficult to accomplish than anticipated due to the formation of secondary disulfide bonds between the newly engineered cysteines and the native ones. Nevertheless, the unpaired cysteines were labeled with 5-(iodoacetamido)fluorescein, and singly labeled species were purified by ion-exchange chromatography. Chymotryptic digestion of the light chains, followed by high performance liquid chromatography separation of the peptides, led to the identification of the fluorescein-labeled cysteines. After light chain exchange into myosin, the position of the thiols was mapped by antifluorescyl antibodies in the electron microscope. Rotary-shadowed images showed the antibody bound at the head/rod junction of myosin for all the mutants. These mapping studies, together with the finding that widely separated cysteines can form multiple disulfide bonds, support a model for LC2 as a flexible, globular molecule that resembles other Ca/Mg-binding proteins in tertiary structure.

Studies on modulation of the effects of colchicine by l-cysteine using bone marrow of Swiss mice

Colchicine (COL) elevates the frequency of micronucleated polychromatic erythrocytes (PE), the ratio of normochromatic to polychromatic erythrocytes (N/PE) and the frequency of large PE due to spindle disruption. Simultaneous i.p. injection of l-cysteine (CYS) does not influence the effects of COL while if administered 1 h prior to COL, CYS suppresses the N/PE ratio and frequency of large PE but not the frequency of micronucleated PE elevated by COL. Preincubation of CYS with COL at 37° C for 1 h results in a significant decrease in all the COL effects. The modulatory effect of exogenous CYS appears to be due to its competition with the endogenous tubulin cysteine residues for interacting with COL.