Of the vulnerability of orphan complex proteins: the case study of the E. coli IscU and IscS proteins.
IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.
Prischi
Filippo
F
MRC National Institute for Medical research, The Ridgeway, London NW7 1AA, UK.
Pastore
Chiara
C
Carroni
Marta
M
Iannuzzi
Clara
C
Adinolfi
Salvatore
S
Temussi
Pierandrea
P
Pastore
Annalisa
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013453:Sulfonylurea Compounds / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D011506:Proteins; D013453:Sulfonylurea Compounds; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine; C058179:sulofenur