The influence of ATP on the association and unfolding of the tyrosine repressor ligand response domain of Haemophilus influenzae.
The secondary structure of the ligand response domain of the Haemophilus influenzae tyrosine repressor, TyrR(lrd), was investigated using CD spectroscopy which revealed 42.5% alpha-helix, 17.6% beta-sheet, and 39.9% loops. Quaternary structure analysis by fluorescence anisotropy showed that TyrR(lrd) is monomeric at a concentration of 100 nM to 2 microM but that the protein readily dimerizes in the presence of its natural ligand ATP. Equilibrium unfolding studies of TyrR(lrd) using guanidinium hydrochloride suggested a two-state model with no detectable stable intermediates. The unfolding transition monitored by CD spectroscopy was responsive to tyrosine and ATP resulting in a shift to higher denaturant concentrations in the presence of these ligands. Differential scanning calorimetry yielded melting temperatures, T(m), of 51.15 and 58.07 degrees C for the unliganded and for the ATP-liganded protein, respectively. ATP is thus proposed to be a major structural cofactor for the molecular architecture of TyrR(lrd).
Kristl
S
S
Institute of Pharmaceutical Chemistry, University of Graz, Universitätsplatz 1, Graz, A-8010, Austria.
Zhao
S
S
Falsone
S F
SF
Somerville
R L
RL
Kungl
A J
AJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019281:Dimerization; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D019791:Guanidine; D006193:Haemophilus influenzae / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D008024:Ligands; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C069256:TyrR protein, E coli; D000255:Adenosine Triphosphate; D019791:Guanidine