Kinetic and proteolytic identification of heat‐induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3‐(3, 4‐dichlorophenyl)‐1, 1‐dimethyl urea (DCMU)‐induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K1) and the relative amount of DCMU‐insensitive electron transport (vmax1/vo) were obtained using a reiterative non‐linear least squares curve‐fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K1 (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol−1. Tryptic susceptibility of a protein region regulating K1 also decreased gradually with exposure to 45°C, suggesting that the heat‐induced increase in K1 might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in vmaxI/vo, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.