Glutamine 53 is a gatekeeper residue in the FK506 binding protein.
Glutamine 53 is a gatekeeper residue in the FK506 binding protein.
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谷氨酰胺 53 是 FK506 结合蛋白中的看门残基。
DOI：
10.1016/s0022-2836(02)00943-9
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发表时间：
2002
影响因子：
5.6
通讯作者：
Logan,TimothyM
中科院分区：
文献类型：
--
作者：
Korepanova,Alla;Douglas,Chanel;Logan,TimothyM
在氨基末端具有三个额外残基的FK506结合蛋白变体（FKBP β）中，研究了尿素未折叠状态下非随机构象平均对折叠途径的影响。三个突变（天冬酰胺，天冬氨酸和苏氨酸）被引入到位置Q53，以增强在尿素未折叠状态下的蛋白质的这一部分中观察到的非天然螺旋形成。NMR分析显示，每个突变体的天然状态下的微小结构变化，但在尿素未折叠状态下观察到每个突变体核的额外中程αN（i，i+2）增强，而不是FKBP构象，表明突变对未折叠状态系综的影响比对天然状态系综的影响更大。等温平衡变性测量结果表明，Q53T和Q53D突变体是不稳定的，而Q53N突变体是稳定的相对于FKBP酶的平衡m值变化不大。Q53N和Q53T的解折叠速率与FKBP的解折叠速率相似，但Q53D的解折叠速率是FKBP的两倍。相比之下，突变对重折叠动力学有更明显的影响。Q53N重折叠稍快，并表现出类似于FKBP的动力学折叠中间体。Q53D和Q53T突变体的重折叠速度也比FKBP β快，但缺乏折叠中间体，表明这些突变体经历了与FKBP β和Q53N不同的折叠轨迹和过渡状态。Q53N、Q53T和Q53D的复性动力学Φ值分别为0.74、1.4和7.9。数据表明Q53在FKBP折叠中充当看门残基。这项研究表明，通过诱变扰动未折叠状态的合奏可以提供洞察残基在折叠途径中发挥重要作用，并代表了一个有吸引力的战略映射的高能量部分的折叠能量景观。
The effect of non-random conformational averaging in the urea-unfolded state on the folding pathway has been investigated in a variant of the FK506 binding protein with three additional residues at the amino terminus (FKBP∗). Three mutations (asparagine, aspartate, and threonine) were introduced into position Q53 to enhance formation of non-native helix observed in this part of the protein in the urea-unfolded state. NMR analysis showed minor structural changes in the native state of each mutant, but additional medium-range αN(i,i+2) of each mutant nuclear Overhauser enhancements were observed in the urea-unfolded state that were not in FKBP∗, indicating that the mutations had a more substantial effect on the unfolded state ensemble than on the native state ensemble. Isothermal equilibrium denaturation measurements showed that the Q53T and Q53D mutants were destabilized, whereas the Q53N mutant was stabilized relative to FKBP∗with little change in the equilibrium m values. The unfolding rates of Q53N and Q53T were similar to that of FKBP∗, but Q53D unfolded twice as fast as FKBP∗. In contrast, the mutations had a more pronounced effect on the refolding kinetics. Q53N refolded slightly faster and exhibited a kinetic folding intermediate similar to that of FKBP∗. The Q53D and Q53T mutants also refolded faster than FKBP∗but lacked the folding intermediate, indicating that these mutants experienced a different folding trajectory and transition state than FKBP∗and Q53N. The refolding kinetic Φ values were 0.74, 1.4 and 7.9 for Q53N, Q53T, and Q53D, respectively. The data point to Q53 functioning as a gatekeeper residue in the folding of FKBP∗. This study shows that perturbing the unfolded state ensemble via mutagenesis can provide insights into residues that play important roles in the folding pathway, and represents an attractive strategy for mapping the high-energy portions of the folding energy landscape.