SUMOylation constitutes a major post-translational modification (PTM) used by 
the eukaryote cellular machinery to modulate protein interactions of the 
targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a 
central and conserved cysteine residue (Cys52) that is located in the 
hydrophobic core of the protein and in tight contact with Phe65, suggesting the 
occurrence of an S/π interaction. To investigate the importance of Cys52 on 
SUMO-1 thermal stability and biochemical properties, we produced by total 
chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide-protein conjugates 
featuring a native isopeptidic bond between SUMO-1 and a peptide derived from 
p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1 
secondary structure and resulted in a dramatic loss of protein thermal 
stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating 
enzyme Upl1 was significantly less efficient than for the wild-type conjugate. 
Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was 
significantly less efficient with the SUMO-1 C52A analog compared to wild-type 
SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1 
conformation and function and the importance of keeping this cysteine intact for 
the study of SUMO-1 protein conjugates.