Divalent ion binding properties of the timothy grass allergen, Phl p 7.
The timothy grass allergen, Phl p 7, was studied by calorimetry, spectroscopy, and analytical ultracentrifugation. As judged by isothermal titration calorimetry (ITC), the protein binds Ca (2+) cooperatively with stepwise macroscopic association constants of 1.73 x 10 (6) and 8.06 x 10 (6) M (-1). By contrast, Mg (2+) binding is sequential with apparent macroscopic association constants of 2.78 x 10 (4) and 170 M (-1). Circular dichroism and ANS fluorescence data suggest that Ca (2+) binding provokes a major conformational change that does not occur upon Mg (2+) binding. Conformational stability was assessed by differential scanning calorimetry (DSC). In phosphate-buffered saline (PBS) containing EDTA, the apoprotein undergoes two-state denaturation with a T m of 78.4 degrees C. In the presence of 0.02 mM Ca (2+), the T m exceeds 120 degrees C. Phl p 7 is known to crystallize as a domain-swapped dimer at low pH. However, analytical ultracentrifugation data indicate that the protein is monomeric in neutral solution at concentrations exceeding 1.0 mM, in both the apo and Ca (2+)-bound states.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA. henzlm@missouri.edu
Davis
Meredith E
ME
Tan
Anmin
A
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D031784:Phleum / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014461:Ultracentrifugation
D000485:Allergens; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; C468525:Phl p 7 allergen; D011522:Protons; D008274:Magnesium; D002118:Calcium