Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein.
Escherichia coli DmsD interacts with the twin-arginine leader sequence of the catalytic sub-unit (DmsA) of DMSO reductase. DmsD was purified as a mixture of a number of different folding forms including: dimer (A); monomer (B); a minor thiol oxidized form; a heterogeneously folded or multi-conformational monomer form which displayed a ladder of bands on native-PAGE (D); and proteolytically degraded and aggregated forms. Polyacrylamide gel electrophoresis (PAGE), under denaturing and non-denaturing conditions, was used to examine the folding and stability of DmsD. Additionally, the biophysical methods of dynamic light scattering, circular dichroism, fluorescence, and mass spectroscopy were also used. Form D could be converted to form B by treatment with 4M urea, which is the concentration at which form B begins to denature. Forms A/B could be converted to D by incubation at pH 5.0. Forms A/B and D all had twin-arginine leader binding activity.
Sarfo
Kwabena J
KJ
Department of Biological Sciences, University of Calgary, 2500 University Dr NW, Calgary, Alta., Canada T2N 1N4.
Winstone
Tara L
TL
Papish
Andriyka L
AL
Howell
Jenika M
JM
Kadir
Hakan
H
Vogel
Hans J
HJ
Turner
Raymond J
RJ
D001120:Arginine / Q000737:chemistry*; D015153:Blotting, Western; D020134:Catalytic Domain; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D008027:Light; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D014508:Urea / Q000494:pharmacology
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D013438:Sulfhydryl Compounds; C408585:twin-arginine translocase complex, E coli; D014508:Urea; D001120:Arginine; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase