3. RESULTADOS Y DISCUSIÓN
3.2. Discusión
3.2.1. Comparación de resultados con el método VAD del proyecto WebRTC
Intrinsic tryptophan fluorescence and l-anilinonaphthalenesulfonate (ANS) binding studies were performed to investigate the conformational changes of Rubisco upon binding and encapsulation by GroEL/ES system.
The changes in the tertiary structure which a protein undergoes upon binding and encapsulation by GroEL can be monitored by observing the intrinsic tryptophan
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fluorescence and by ANS binding studies. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine. Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ~ 300 to 350 nm depending on the polarity of the local environment. ANS is a probe for apolar binding sites whose fluorescence is strongly dependent on the hydrophobicity of the environment. ANS accumulates in the solvated hydrophobic core of early folding intermediates generally termed ‘molten globule’. GroEL-stabilized proteins show strong ANS fluorescence (Martin et al., 1991).
Rubisco large subunit from Rhodospirillum rubrum (Rr-RbcL) contains six tryptophan residues and that of Synechococcus sp. PCC6301 (Syn6301-RbcL) contains nine. The GroEL and GroES lack any tryptophans (Hemmingsen et al., 1988; Hohn et al., 1979). Single ring version of GroEL (SR-EL) was used for the ANS experiments (Hayer- Hartl et al., 1996; Weissman et al., 1995). SR-EL binds GroES in an ATP-dependent manner, but is unable to dissociate it due to the absence of an allosteric signal from the Gro-EL trans-ring. Thus GroES is believed to stably encapsulate protein substrate in the SR-EL/GroES complex without the possibility of active unfolding (Hayer-Hartl et al., 1996).
Native Rr-RbcL showed a maximum emission of tryptophan fluorescence at 335 nm (Figure 27A, black curve), while guanidium denatured Rubisco showed the maximum emission of tryptophan fluorescence at 365 nm (Figure 27A, pink curve) accompanied by a remarkable decrease in the intensity of fluorescence. GroEL-bound Rr-RbcL showed an emission maximum at 348 nm (Figure 27A, green curve & blue curve), i.e. ~57% shift from the denatured to the folded state. High ANS-fluorescence was observed when the unfolded Rr-RbcL was stabilized by SR-GroEL, indicating a massive binding of ANS to the hydrophobic surfaces of the early folding intermediates, in a conformation lacking ordered tertiary structure (molten globule state) (Figure 27B).
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Upon addition of GroEL/ES and Mg-ATP to the unfolded Rr-RbcL, the tryptophan fluorescence emission maximum shifted back to 335 nm, identical to the emission maximum of the native protein (Figure 27A, red curve). This indicates the folding and reconstitution of Rr-RbcL dimer. This was further supported by the decrease in the intensity of ANS fluorescence observed during the refolding reaction (Figure 27B).
Figure 27. Tryptophan and ANS fluorescence studies of Rr-RbcL upon interaction with GroEL
A. Tryptophan fluorescence in RbcL refolding: Denatured Rr-RbcL2 was diluted 100-fold (0.25 μM RbcL
monomer) into ice-cold assay-buffer containing 0.5 μM GroEL. After incubation for 10 min on ice, tryptophan fluorescence (excitation 295 nm, emission scan 315-450 nm) was measured. Then 1 μM GroES was added and tryptophan fluorescence was measured again. The reaction was supplemented with 2 mM ATP and refolding kinetics was observed by monitoring the change in tryptophan fluorescence over time. A tryptophan scan at the end of refolding was measured. If only native or denatured substrate had to be analyzed, reactions were modified accordingly. Background fluorescence of chemically identical reactions lacking RbcL was subtracted.
B. ANS-fluorescence in RbcL refolding: Denatured Rr-RbcL2 was diluted 100-fold (0.25 μM RbcL monomer)
into ice-cold assay-buffer 1 containing 1 μM ANS, 1 μM SR-GroEL and 2 μM GroES. After incubation for 10 min on ice, ANS fluorescence (excitation 390 nm, emission scan 420-550 nm) was measured. Refolding was started with the addition of 2 mM ATP and the kinetics was observed by monitoring the change in ANS fluorescence over time. At the end of refolding, an ANS fluorescence scan was taken. If only native or denatured substrate had to be analyzed, reactions were modified accordingly. Data were corrected for background fluorescence of chemically identical reactions lacking RbcL and emission at 470 nm was depicted.
Native Syn6301-RbcL8 showed a fluorescence maximum at 355 nm (Figure 28A,
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decrease in the emission intensity of more than 40%. The emission maximum of GroEL bound Syn6301-RbcL was 355 nm, identical to that of native protein, but fluorescence intensity was slightly lower (Figure 28A, green curve). Similar to Rr-RbcL experiments, high ANS-fluorescence was observed when the unfolded Syn6301-RbcL was stabilized by SR-GroEL (Figure 28B).
Upon addition of GroEL/ES alone or together with Mg-ATP to the unfolded Syn6301-RbcL, the tryptophan fluorescence emission maximum was retained at 355 nm, identical to the emission maximum for native protein with a further decrease in the intensity (Figure 28A, blue curve & red curve). This conformation probably reflects the monomeric folded state of RbcL. The decrease in the ANS-fluorescence again confirms the changes in the tertiary or quaternary structure of the enzyme molecule during the refolding reaction (Figure 28B).
Figure 28. Tryptophan and ANS fluorescence studies of Syn6301-RbcL upon interaction with GroEL
A. Tryptophan fluorescence in RbcL refolding: Denatured Syn6301-RbcL8 was diluted 100-fold (0.25 μM
RbcL monomer) into ice-cold assay-buffer containing 0.5 μM GroEL. After incubation for 10 min on ice, tryptophan fluorescence (excitation 295 nm, emission scan 315-450 nm) was measured. Then 1 μM GroES was added and tryptophan fluorescence was measured again. The reaction was supplemented with 2 mM ATP and refolding kinetics was observed by monitoring the change in tryptophan fluorescence over time. A tryptophan scan at the end of refolding was measured. If only native or denatured substrate had to be analyzed, reactions were modified accordingly. Background fluorescence of chemically identical reactions lacking RbcL was subtracted.
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B. ANS-fluorescence in RbcL refolding: Denatured Syn6301-RbcL8 was diluted 100-fold (0.25 μM RbcL
monomer) into ice-cold assay-buffer 1 containing 1 μM ANS, 1 μM SR-GroEL and 2 μM GroES. After incubation for 10 min on ice, ANS fluorescence (excitation 390 nm, emission scan 420-550 nm) was measured. Refolding was started with the addition of 2 mM ATP and the kinetics was observed by monitoring the change in ANS fluorescence over time. At the end of refolding, an ANS fluorescence scan was taken. If only native or denatured substrate had to be analyzed, reactions were modified accordingly. Data were corrected for background fluorescence of chemically identical reactions lacking RbcL and emission at 470 nm was depicted.