國家衛生研究院 NHRI:Item 3990099045/6696
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    題名: High-order correlated motion in single-molecule human adenylate kinase 1
    作者: Lin, CY;Huang, JY;Lo, LW
    貢獻者: Division of Medical Engineering Research
    摘要: The growing body of data shows protein motion plays an important role in the catalytic processes of enzymes. Human adenylate kinase 1 (AK1) is the key enzyme in maintaining the cellular energy homeostasis. The conformational change of AK1 involves large-amplitude rearrangements of the enzyme's lid domain. Observing a single molecule can remove the ensemble average, thus allows the exploration of hidden structural heterogeneity. To observe the enzyme actions of AK1 at the single-molecule level, we developed a data-taking scheme allowing us to observe the single-molecule kinetics in long time without the limitation by photobleaching. We labeled the core domain and the rim of AK with two Alexa-532 chromophores. Due to the self quenching from photo-induced electron transfer, the conformation changes of the core domain can be accurately encoded into the fluorescent intensity traces with a spatial sensitivity <1nm. Because Mg2+ ion is known to be an affecter in the energy signaling network in cells, we first investigated the effect of Mg2+ on the catalytic kinetics of AK. By analyzing the photon traces, we found that the binding of Mg2+ ions to arginines and lysines of AK increases the structural heterogeneity, which then couples to the core domain and changes the catalytic function. We further discovered that ATP/AMP substrate binding suppresses the conformation fluctuation of AK and improves the thermal stability of the core domain. The resulting third-order correlation functions of photon traces do not have time-reversal symmetry, indicating single-molecule AK is in a nonequilibrium steady state. We employed hidden Markov model with Baysian inference to verify that the photon traces are originated from transitions among three states (open, mid, close). Substrate binding to AK causes different domains of AK to move in a sequential order and suppresses the thermally-activated random motions.
    日期: 2012-01
    關聯: Biophysical Journal. 2012 Jan;102(3, Suppl. 1):272a.
    Link to: http://dx.doi.org/10.1016/j.bpj.2011.11.1498
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0006-3495&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000321561201680
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