Holophytochrome Assembly

1. The formation of the fluorescent PEB-

phytochrome adduct is initiated in a semimicrofluorescence cuvette by addition of apophytochrome (10 nM final concentration) from a concentrated stock solution to a greater-than or equal to 70-fold molar excess of PEB (typically 0.5 to 15 pM final concentration) in TEGE buffer [10 mM Tris-HCl, pH 8.0, 25% (vol/vol) ethylene glycol, 1

mM EDTA, 1 mM PMSF, and 1 mM DTT]. The PEB is dissolved in Me2SO, with the final Me2SO concentration in the assay no greater than 2% (vol/vol). The assay mixture is rapidly mixed and placed in a fluorescence spectropho-tometer. Typically, measurements are performed with samples maintained at room temperature (22°-25°C).

Scheme 1. Kinetic analysis of holophytochrome assembly (adapted from Reference 39). The enzyme reaction of a bilin, typically PEB, with apoPC to produce a fluorescent product is shown in the diagram. Equation 1 is the integrated rate equation describing this reaction. Equation 2 defines the kinetics of bilin—apophytochrome adduct formation. If the bilin precursor concentration is kept essentially constant, by providing a large excess, semilog plots of the fraction of apophytochrome remaining are expected to be linear as described in Equation 3. Equation 4 and its reciprocal, Equation 5, provide a means for determining the affinity of apophytochrome for a particular bilin, Knin, and the rate constant, or turnover, of apophytochrome for a particular bilin,

2. For time-based measurements, samples were excited with 570 nm light with 2 nm bandpass. Fluorescence emission data was collected at 586 nm with 16 nm bandpass. Data was collected with 1-second integration for 15 to 30 minutes. Saturated fluorescence intensity (i.e., 100% assembly) is determined in parallel by incubating a control sample of phytochrome with a very large excess of PEB (>500-fold molar excess) for more than 1 hour.

3. The equations outlining the analysis of data for the standard kinetic assay with PEB and apophytochrome are outlined in Scheme 1, and example data is shown in Figure 3. Raw fluorescence data is transformed using Equation 3 in Scheme 1. When data is replotted on a semilog graph, kapp values for each assembly reaction are determined from the slope of the line. According to Equation 5 (Scheme 1), 1/kapp values for the different assemblies are then plotted versus 1/(PEB). The x- and y-intercepts for this data provide the Kbilin and kcat, or k2, respectively, for PEB.

Variations:

4. The analysis of data for the competitive assay using a reversible inhibitor of PEB-phytochrome formation such as BV (see Scheme 2), is carried out in much the same manner as outlined for the standard assay above. The raw fluorescence data is transformed using Equation 8 (Scheme 2). This data is graphed on a semilog plot to obtain the kapp as before. The K for BV, or KBv, is estimated using the x-intercept of the plot of the 1/kapp versus the BV concentration (Equation 10 in Scheme 2).

5. The analysis of the data when using an irreversible inhibitor of PEB-apophy-tochrome adduct formation, such as POB or PCB, is much different from the previous examples (Scheme 3).

Experimentally, the amount of competitor bilin is estimated from the degree of fluorescence inhibition relative to the control reaction with no inhibitor. Since both the concentration of PEB-phytochrome (PC) adduct and the kapp for PEB-PC formation are known, the kIapp can be calculated using Equation 13 (Scheme 3). The Kjbilin is obtained following a double reciprocal plot of the kIapp versus the bilin concentration as shown in Equation 15 (Scheme 3).

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