KINETICS AND MECHANISM OF HEME-INDUCED REFOLDING OF HUMAN ALPHA-GLOBIN
KINETICS AND MECHANISM OF HEME-INDUCED REFOLDING OF HUMAN ALPHA-GLOBIN
复制标题
DOI：
10.1073/pnas.78.2.780
复制
发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
BEYCHOK, S
中科院分区：
文献类型：
--
作者：
LEUTZINGER, Y;BEYCHOK, S
Hb α和β链是紧密堆积的、高度（75%）螺旋稳定的分子。血红素的去除导致未折叠的（30%螺旋）不稳定的珠蛋白链，其可以通过与血红素重组而重新折叠成天然构象。通过3种停流技术研究血红素结合的动力学和随后的构象变化：荧光猝灭，其监测结合的血红素和A12（14）α之间的空间取向和距离。色氨酸; Soret带最大吸收，其位置和强度取决于血红素的局部环境和轴向配体的性质;以及远紫外圆二色性[CD]，其直接测量二级结构的恢复。荧光猝灭是双相的。初始的二级衰减，代表总振幅的80-85%，标志着氯化血红素二氰化物以3.3 × 104的速率常数结合到相对明确的位点。107 M-1 s-1，对应于在2.4 μ M反应物下10 ms的半衰期。Soret吸收和CD也是多相的，所有3个探针检测到半衰期为25-40 s的一级过程，在此期间建立了血红素口袋的最终二级和三级结构，并且血红素和A12色氨酸之间的空间关系是固定的。代表总骨架重折叠的2/3的较慢CD变化，半衰期为116 s，标志着天然亚基构象的完全获得。血红素口袋的残基在整个链折叠之前就达到或接近其最终的三维结构。这些测量代表了辅基诱导的二级结构形成的速率的直接观察，并说明了多探针分析在概述蛋白质折叠途径中的优势。
Hb .alpha. and .beta. chains are tightly packed, highly (75%) helical stable molecules. Removal of the heme results in unfolded (30% helical) unstable globin chains that can be refolded to the native conformation by recombination with heme. The kinetics of heme binding and the ensuing conformational changes were studied by 3 stopped-flow techniques: fluorescence quenching, which monitors the spatial orientation and distance between the bound heme and the A12(14).alpha. tryptophan; absorption at the Soret band maxima, whose position and intensity depend on the local environment of the heme and nature of the axial ligands; and far-UV circular dichroism [CD], which directly gauges the recovery of secondary structure. The fluorescence quenching was biphasic. An initial 2nd-order decay, representing 80-85% of the total amplitude, marked the binding of hemin dicyanide to a relatively well-defined site at a rate constant of 3.3 .times. 107 M-1 s-1, corresponding to a half-time of 10 ms at 2.4 .mu.M reactants. The Soret absorption and CD were also multiphasic, all 3 probes detecting a 1st-order process of half-time 25-40 s, during which the final secondary and tertiary structures of the heme pocket were established, and the spatial relationship between the heme and the A12 tryptophan was fixed. A slower CD change representing 2/3 of the total backbone refolding, with a half-time of 116 s, marked the full acquisition of the native subunit conformation. The residues of the heme pocket achieve or closely approach their final 3-dimensional structure well before the entire chain is folded. These measurements represent a direct observation of the rate of prosthetic group-induced secondary structure formation and illustrate the advantages of multiple probe analysis in outlining a protein folding pathway.