Roles of cysteine residue on chimeric green fluorescent protein: implications on protein solubilization and fluorescent property
Cysteine (a sulfur-based amino acid) has widely been used to aid the biomolecular structure investigation, protein purification, and metal remediation. However, major drawbacks from the difficulty to maintain its reduced state and the tendency to form protein multimerization become obstacles of its applicability. In the present study, significant roles of cysteine residue on the alterations of intrinsic biological activity as well as the expression and localization of chimeric green fluorescent protein (GFP) have been investigated. A series of chimeric GFPs carrying a helical conformation of cysteine-rich peptides (designated as (CXXX)3GFPuv and (CX)3P(HX)3GFPuv) was successfully constructed. The presence of cysteine residues significantly exerted some suppressing effect on the fluorescent emission at both the cellular and protein levels. In addition, the majority of proteins (>95%) was found to be aggregated in the debris fraction. By contrarily, substitution of cysteine with histidine residue rendered the proteins to be more soluble in the cytoplasmic portion. More importantly, enhancement of fluorescent activity up to 2 folds could be detected in the case of chimeric (HXXX)3GFPuv and (HX)3P(HX)3GFPuv. Such prominent effects were experimentally proven to be attributable to the disulfide bond formation. Recovery of both metal-binding capability and the fluorescent activity was more pronounced in the presence of reducing agent. Conclusion can be drawn that the presence of cysteine residue on the biological macromolecules may influence their solubility and functions while exhibiting diverse characteristics in the oxidative and reductive situations. Further investigations, particularly on the use of computational analysis and quantum mechanics, are needed to be performed to gain more understanding on the underlying mechanisms of cysteine in biological system.
Chartchalerm Isarankura-Na-Ayudhya, Chanin Nantasenamat, Tanawut Tantimongcolwat, Apilak Worachartcheewan, Sakda Yainoy, Thummaruk Suksrichavalit, and Virapong Prachayasittikul. "Roles of cysteine residue on chimeric green fluorescent protein: implications on protein solubilization and fluorescent property" European Journal of Scientific Research 26 (2009): 578-595.