Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry.
Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.
Potier
N
N
Department of Physics, University of Manitoba, Winnipeg, Canada.
Donald
L J
LJ
Chernushevich
I
I
Ayed
A
A
Ens
W
W
Arrowsmith
C H
CH
Standing
K G
KG
Duckworth
H W
HW
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004355:Drug Stability; D009682:Magnetic Resonance Spectroscopy; D009876:Operon; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins