Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118.
In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118. From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama 700-8530, Japan.
J
J
Futami
H
H
Tada
M
M
Seno
S
S
Ishikami
H
H
Yamada
D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010179:Pancreas / Q000737:chemistry; D017510:Protein Folding; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D012260:Ribonucleases; D014357:Trypsin; D003545:Cysteine