N-terminal extension changes the folding mechanism of the FK506-binding protein.
N-terminal extension changes the folding mechanism of the FK506-binding protein.
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N 端延伸改变了 FK506 结合蛋白的折叠机制。
DOI：
10.1110/ps.14801
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Logan,TM
中科院分区：
文献类型：
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作者：
Korepanova,A;Douglas,C;Leyngold,I;Logan,TM
许多用于提高重组蛋白产量的蛋白融合系统导致在所需蛋白上添加少量氨基酸残基。在这里，我们研究了短（三个氨基酸）N末端延伸对FK 506结合蛋白（FKBP）的平衡变性和动力学折叠和解折叠反应的影响，并将所得结果与缺乏该延伸的FKBP变体的数据进行了比较。等温平衡变性实验表明，N末端延伸具有轻微的不稳定效应。NMR研究表明，N-末端延伸轻微扰乱了延伸位点附近的蛋白质结构，影响较小，传播到FKBP的单个α-螺旋中。这些结构扰动可能是微分稳定性的原因。与相对较小的平衡效应相反，N末端延伸产生了在较短结构中未观察到的动力学折叠中间体。在延伸中具有不同氨基酸序列的构建体上进行的动力学实验表明，延伸的长度和序列都有助于观察到的平衡和动力学效应。这些结果指出N末端在FKBP折叠中的重要作用，并表明在许多真核和原核蛋白中观察到的N末端甲硫氨酸去除的生物学后果是增加多肽链的折叠效率。
Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N‐terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506‐binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N‐terminal extension had a slight destabilizing effect. NMR investigations showed that the N‐terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single α‐helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N‐terminal extension generated a kinetic‐folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N‐terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.