Studies of macromolecular crowding

在与E. Haas小组的合作中，我们研究了一种小的“惰性”co溶质三甲胺氧化物对腺苷酸激酶的构象异构化的影响，这种酶在催化循环中会发生显著的构象变化。底物核苷酸（AMP、ADP、ATP或其类似物）的结合伴随着由含有配体结合位点的间隙闭合引起的压实。构象变化是通过时间依赖性荧光共振能量转移（FRET）的供体和受体之间的荧光基团放置在选定的位置沿着肽链通过位点特异性诱变表征。迄今为止获得的结果分析表明，相对于不太紧密或“开放”的构象，足够浓度的氧化三甲胺稳定紧密或“封闭”的构象，导致酶对配体的亲和力大幅增加。最近发表了一份关于这项工作的报告。

In collaboration with the group of E. Haas we have investigated the effect of a small "inert" cosolute, trimethylamine oxide, on the conformational isomerization of an enzyme, adenylate kinase, that is known to undergo significant conformational changes during its catalytic cycle. Binding of substrate nucleotides (AMP, ADP, ATP, or analogs thereof) is accompanied by compaction resulting from the closure of clefts containing ligand binding sites. Conformational changes are being characterized via time-dependent fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores placed at selected locations along the peptide chain through site-specific mutagenesis. Analysis of the results obtained to date indicate that sufficient concentrations of TMAO stabilize compact or "closed" conformations relative to less compact or "open" conformations, leading to a substantial increase in the affinity of the enzyme for ligands. A report of this work was recently published.