Molecular topography of phytochrome as deduced from the tritium-exchange method

Abstract

The hydrogen-tritium-exchange measurements on phytochrome have been performed to detect the conformational differences between the red-absorbing (Pr) and the far-red absorbing (Pfr) forms of phytochrome. The large and small Pfr molecules revealed more exchangeable protons that did the corresponding Pr molecules by 96 and 70 protons, respectively. These results suggest that the Pr leads to Pfr phototransformation is accompanied by an additional exposure of the peptide chains in the Pfr molecule. Of 1682 theoretically exchangeable hydrogens in undegraded phytochrome, only 442 (26%) and 346 (21%) protons were found to be exchangeable (excluding instantaneously exchangeable protons that cannot be determined by the present method). Thus, the phytochrome protein appears to be compact and highly folded. The kinetic analyses of the tritium exchange-out curves indicate that two kinetically different groups are responsible for the conformational differences between the Pr and Pfr forms of phytochrome. These components are due to (1) the exposure of hydrogen-bonded peptide segments (alpha helix and/or beta-pleated sheet) in the chromophore vicinity of Pfr and (2) the exposure of hydrogen-bonded peptide segments on the chromophore peptide domain as well as on the chromophore-free tryptic domain of undegraded phytochrome.

Molecular topography of intact phytochrome probed by hydrogen-tritium exchange measurements

“…In this study, we discuss the protein structure of intact phytochrome by employing the hydrogen-tritium exchange method. From the comparison of the present data with our previous H-T exchange data on the degraded phytochromes (Hahn & Song, 1982), we wish to elucidate the molecular topography of intact phytochrome in this paper.…”

“…The gel permeation chromatography developed by Englander (1963Englander ( , 1968 was employed to follow both the exchange-in and exchange-out rates of hydrogen-tritium exchange of the intact phytochrome, as described previously (Hahn & Song, 1982). In exchange-in experiments, 1,5 mL of phytochrome (A660 ~0.2) in 0.1 M potassium phosphate buffer, pH 7.8, containing 5 mM EDTA, 14 mM 2-ME, and 5% glycerol was incubated with 30 nL of tritiated water (specific activity 500 mCi/g).…”

“…The kinetics of exchange out were measured by both onecolumn and two-column methods as described previously (Hahn & Song, 1982). The one-column method was used for initial exchange-out kinetics within 30 min, and the twocolumn procedure was employed for the exchange-out measurements of longer incubation times.…”

“…In order to deduce the molecular topography of oat phytochrome, the hydrogen-tritium exchange kinetics of intact phytochrome (molecular weight 124000) have been measured and compared with the data obtained previously with degraded proteins [Hahn, T.-R., & Song, P.-S. (1982) Biochemistry 21, 1394-1399], In comparison with the large (molecular weight 118 000) and small (molecular weight 60000) phytochromes, the intact phytochrome revealed a smaller difference in the number of exchangeable protons between the red-absorbing form of phytochrome (Pr) (368 protons per molecule) and the far-red-absorbing form of phytochrome (Pfr) (406). In addition, the intact Pr exchanged 22 more protons than the large Pr (346), whereas the intact Pfr exchanged 36 less protons than the large Pfr (442), indicating that an additional surface area exposed in the large Pfr is shielded in the intact Pfr.…”

“…This method is particularly useful to detect conformational changes of proteins, because no critical alteration of the protein structure is induced by the method itself. The structural differences between the degraded Pr and Pfr forms of phytochrome have been determined by the method of Hahn & Song (1982).…”