| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥10,200,000 (Direct Cost: ¥10,200,000)
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| Research Abstract |
The objective of the present study is to elucidate the molecular mechanism of functional expression of the Escherichia coli chaperonin GroEL/ES. For this purpose, we investigated the interactions of the chaperonin with folding intermediates of three globular proteins, α-lactalbumin (αLA), staphylococcal nuclease (SNase) and green fluorescent protein (GFP), which have very different physical properties. We also investigated physicochemically the interactions of GroEL with the nucleotides (ATP and ADP) that are indispensable for the chaperonin function and the analogs of the nucleotides (ATPγS and AMP-PNP). The following results were obtained.
(1) We used an SNase mutant, in which the refolding kinetics were significantly simplified, and apo-αLA that showed a simple single relaxation kinetics of refolding, as model target proteins of GroEL. We studied the effect of the chaperonin on the refolding kinetics of these target proteins by stopped-flow fluorescence spectroscopy. Especially for α
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LA, we succeeded the quantitative analysis of the reaction curves by computer simulations. When we added a nucleotide in the absence of GroES, only ATP was effective for reducing the affinity of GroEL for the target protein. However, when GroES was present, not only ATP but also the ATP analogs were found to effectively reduce the GroEL affinity for the target protein.
(2) We constructed E. coli expression systems for GFP and its Cycle3 mutant and studied their in vitro refolding reactions by fluorescence spectroscopy. These proteins can also be used as model target proteins of the chaperonin.
(3) We studied the interactions of GroEL with ADP and the ATP analog by titration calorimetry and fluorescence spectroscopy. In the latter method, we used the fluorescence intensity change of pyrenyl GroEL to monitor the interactions. We found that these nucleotides bound to GroEL in a non-cooperative manner and that there were two kinds of the binding sites with different affinities. Only ATP induced a cooperative fluorescence change of pyrenyl GroEL., suggesting that ATP hydrolysis was required for the cooperative change. From this results together with the above results in(1), the ATP hydrolysis and the resultant cooperative change are expected to by required for releasing the target protein form GroEL. Less
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