Optical detection of disordered water within a protein cavity.
Optical detection of disordered water within a protein cavity.
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DOI：
10.1021/ja903409j
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发表时间：
2009-09-02
影响因子：
15
通讯作者：
Esquerra, Raymond M.
中科院分区：
文献类型：
--
作者：
Goldbeck, Robert A.;Pillsbury, Marlisa L.;Jensen, Russell A.;Mendoza, Juan L.;Nguyen, Rosa L.;Olson, John S.;Soman, Jayashree;Kliger, David S.;Esquerra, Raymond M.
内部水分子对蛋白质结构和功能很重要，但位置紊乱和低占有率可能会妨碍 X 射线晶体学的检测。在这里，我们表明，即使在相应的晶体结构中不易观察到溶剂的存在，也可以通过五配位血红素吸收光谱的细微变化来检测肌红蛋白突变体远端空腔内的水。在野生型 (wt) 脱氧肌红蛋白晶体结构的远端血红素袋内可以看到与远端组氨酸 (His64) 氢键结合的明确的非配位水分子。这种水的置换降低了配体进入wt Mb的速率，并且我们之前已经表明，在MbCO复合物的激光光解之后，很容易通过光学方法检测到这种水的进入。然而，对于 L29F 和 V68L Mb，尽管 His64 仍然存在，但在晶体结构的电子密度图中没有看到溶剂分子的离散位置，并且观察到表明内部有水的配体结合速率较慢。相比之下，激光光解后产生的 L29F 和 V68L deoxyMb 可见吸收带的时间分辨扰动检测到这些变体远端口袋内水的进入和显着占据。因此，五坐标血红素的光谱扰动为测量血红素蛋白活性位点的非特异性水合提供了一个潜在的稳健系统。
Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by x-ray crystallography. Here we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well defined, non-coordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring non-specific hydration of the active sites of heme proteins.
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DOI：
10.1107/s090744499900935x
发表时间：
1999-10-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Pflugrath, JW
通讯作者：
Pflugrath, JW
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
4.8
作者：
Dantsker, D;Roche, C;Friedman, JM
通讯作者：
Friedman, JM
DOI：
10.1073/pnas.0803764105
发表时间：
2008-06-24
影响因子：
11.1
作者：
Ishikawa, Haruto;Kwak, Kyungwon;Fayer, Michael D.
通讯作者：
Fayer, Michael D.
影响因子：
15
作者：
Hirota, S;Li, TS;Kitagawa, T
通讯作者：
Kitagawa, T