Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

Abstract

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.

Figure 1.

Time dependence of the IR difference spectrum of GroEL (left panel) and SR1 (right panel) in the presence of ATP. Measurements were performed after release of 0.8 mM ATP in samples containing 0.8 mM protein subunits, at 25°C in H₂Obased buffer. Spectra were recorded at the indicated times after the photolysis flash. Average of 10 (GroEL) and 11 (SR1) independent experiments. For the sake of comparison, the photolysis spectrum is also shown (bottom traces).

Figure 2.

Kinetics of selected infrared absorbance changes of GroEL and SR1 induced by ATP. (A) Time dependence of the absorbance changes at 1570 (circles) and 1560 (squares) cm⁻¹ for GroEL (filled symbols) and SR1 (open symbols). (B) Time dependence of the intensity of differential signals at 1655 (squares) and 1268 cm⁻¹ (triangles). Different constants were added to the values obtained for GroEL (filled symbols) and SR1 (open symbols) to allow for a simultaneous representation. Spectra were recorded in H₂O-based buffer as described in Figure1 ▶.

Figure 3.

(A) Time dependence of the absorbance changes at 1655 (circles) and 1568 (squares) cm⁻¹ for GroEL (filled symbols) and SR1 (open symbols). The nucleotide/protein subunit molar ratio was 1 in both cases. (B) Kinetics of the 1568 cm⁻¹ differential signal obtained at nucleotide/wild-type GroEL subunit molar ratios of 0.5 (filled squares) and 2 (open circles). Other details are as in Figure2 ▶. The fluorescence changes of pyrenyl-GroEL in the presence of ATP (nucleotide/GroEL subunit molar ratio 1 [A] and 0.5 [B]) are shown as solid lines (Kad et al. 1998).

Figure 4.

ATP-induced IR absorbance changes in D₂O medium. The difference spectra were recorded 3 sec after release of 1.6 mM ATP in samples containing 0.8 mM protein subunits. Wild-type GroEL (thick solid line), E461K (dashed/dotted line), E434K (dotted line), and SR1 (thin solid line). Photolysis reaction recorded under identical experimental conditions but in the absence of proteins (○). Data are the average of 15 (GroEL), 10 (E461K), 12 (E434K), and 16 (SR1) different experiments. Other details are as in Figure1 ▶. Due to the higher intrinsic ATPase activity of mutants E461K and E434K (Sot et al. 2002), the amount of ATP released in these samples was 1.6 mM to extend the binding event to 3 sec. (Inset) Difference spectra obtained after subtracting the photolysis spectrum from those obtained in the presence of the proteins. The criterion used to choose the subtraction factor was to cancel the negative signal at 1526 cm⁻¹, indicated by an arrow.

Figure 5.

ADP-induced absorbance changes in D₂O medium. Difference spectra recorded 3 sec after the release of ~0.8 mM ADP into samples containing 0.8 mM protein subunits. Wild-type GroEL (thick solid line), E461K (dashed/dotted line), E434K (dotted line), and SR1 (thin solid line). Photolysis spectrum (○). Average of seven (GroEL), eight (E461K), five (E434K), and eight (SR1) experiments.