研究人員對普遍存在的運動蛋白kinesin已經進行了廣泛研究，但一個基本的機制問題仍然沒有得到回答：當kinesin在每8納米的步長之間等待時它是兩頭都與微管結合在一起的還是只有一頭與微管結合？現在，Mori等人研製出單分子螢光共振能量轉移傳感器（smFRET），用來在該運動蛋白沿微管運動時對其進行跟蹤。他們發現，在生理濃度的ATP中，kinesin是以一種雙頭結合狀態在兩個步長之間等待的，而在低濃度的ATP中，它主要以單頭結合狀態出現。
原始出處:
Nature 450, 750-754 (29 November 2007) | doi:10.1038/nature06346; Received 18 April 2007; Accepted 2 October 2007; Published online 14 November 2007
Teppei Mori1, Ronald D. Vale2 & Michio Tomishige1,2
Correspondence to: Ronald D. Vale2 Correspondence and requests for materials should be addressed to R.D.V. (Email: vale@cmp.ucsf.edu).
Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules1, 2 by hydrolysing ATP3 and moving processively in 8-nm steps4. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads')5, 6, 7, a process driven by a conformational change in the neck-linker domain of kinesin8, 9, 10, 11, 12. However, the 'waiting conformation' of kinesin between steps remains controversial13, 14, 15, 16—some models propose that kinesin adopts a one-head-bound intermediate17, 18, 19, 20, 21, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits7, 22, 23. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.
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