The chemical synthesis of the GstI protein by NCL on a X-Met site.
The small GstI protein (63 amino acids) of Rhizobium leguminosarum is the endogenous inhibitor of the glnII (glutamine synthetase II) gene expression. It has been suggested that GstI has a predominantly beta-structure and mediates the block of translation and stabilization of glnII mRNA through direct binding to its 5' untranslated region. Because of the unavailability of adequate amounts of purified recombinant protein, the mechanism as well as the protein tridimensional structure remain very poorly understood. To obtain the full-length protein, we have undertaken the chemical synthesis of the protein by different approaches. In a first attempt, the stepwise synthesis was unsuccessful, with strong aggregation experienced on the N-terminal side, after residue 44 from the C-terminus. In a second approach, we set up the conditions to carry out a native chemical ligation (NCL). Albeit the protein contains two Cysteine residues, located at positions 40 and 47, to minimize the size of the N-terminal segment to be synthesized, we have devised an alternative strategy of ligation on Met32, utilizing homoCys as the ligating moiety and then alkylating the resulting polypeptide with methyl iodide. New conditions to quantitatively methylate thiol groups in complex polypeptides have been conceived, obtaining the protein in very good yields and purity. A CD spectroscopy investigation has revealed that the protein does not adopt canonical secondary structures but is very rich in beta-structure (approximately 60%), in agreement with a previous study carried out on samples obtained by recombinant methods.
Saporito
Angela
A
Istituto di Biostrutture e Bioimmagini del CNR, Sezione Biostrutture, NAPOLI, via Mezzocannone 16, 80134 Napoli, Italy.
Marasco
Daniela
D
Chambery
Angela
A
Botti
Paolo
P
Monti
Simona M
SM
Pedone
Carlo
C
Ruvo
Menotti
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D006710:Homocysteine / Q000378:metabolism; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000138:chemical synthesis* / Q000737:chemistry
D001426:Bacterial Proteins; C414318:GstI protein, Rhizobium leguminosarum; D012097:Repressor Proteins; D006710:Homocysteine; D008715:Methionine; D003545:Cysteine