In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.
A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.
Williams
Neal K
NK
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Prosselkov
Pavel
P
Liepinsh
Edvards
E
Line
Inara
I
Sharipo
Anatoly
A
Littler
Dene R
DR
Curmi
Paul M G
PM
Otting
Gottfried
G
Dixon
Nicholas E
NE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D000458:Cyanobacteria / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D001426:Bacterial Proteins; D011506:Proteins; D011994:Recombinant Proteins; D006859:Hydrogen; D014357:Trypsin; D004265:DNA Helicases; D053488:DnaB Helicases