Small-angle X-ray scattering studies on the macromolecular structure of the red-light-absorbing form of 121 kDa pea phytochrome and its 114 kDa chromopeptide

Abstract

The macromolecular structure of 121 kDa pea phytochrome and its 114 kDa chromopeptide was studied by small-angle X-ray scattering (SAXS) and steric exclusion column chromatography. The molecular mass of the 114 kDa chromopeptide in the red-light-absorbing form ( P r) was determined to be 228 kDa in 100 mM potassium phosphate and 1 mM EDTA (pH 7.8) by SAXS, indicating that the chromopeptide is a dimer. The radius of gyration ( R g) of the chromopeptide was determined to be 53.8 Å by Guinier analysis of SAXS. The molecular mass of a sphere with an R g of 53.8 Å is calculated to be 1140 kDa, which is far greater than the observed molecular mass for the 114 kDa chromopeptide. The discrepancy comes from the deviation of the molecular shape from a sphere and/or the dense distribution of electrons in the marginal area of the molecule. The apparent molecular mass of the chromopeptide was determined to be 314–318 kDa from steric exclusion column chromatography, indicating a nonglobular molecular shape. Assuming the overall shape is an ellipsoid of revolution, a prolate one ( a = 24, b = 115 A ̊ and an oblate one ( a = 85, b = 9 A ̊ ), where a and b are the equatorial diameter and the half-axial length, respectively, are calculated to have an R g of 53.8 Å and a molecular mass of 228 kDa. The R g of the 121 kDa phytochrome was 54.0 Å, suggesting that the terminal polypeptide(s), which is iacking in the 114 kDa chromopeptide, contributes only slightly to the molecular dimension. The apparent molecular mass of the chromopeptide increased to 321–324 kDa after red-light irradiation, indicating that the far-red-light-absorbing form ( P fr) of the chromopeptide has a different molecular dimension from that of P r. The Guinier analysis of the SAXS of the chromopeptide in P fr, however, was not successful, owing to the low content of P fr and the aggregation after saturating red-light irradiation.