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
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
J Am Chem Soc. 2009 Sep 2;131(34):12265-72. doi: 10.1021/ja903409j.
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, noncoordinated 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 nonspecific hydration of the active sites of heme proteins.
内部水分子对蛋白质的结构和功能很重要，但位置无序和占有率低会使其难以通过X射线晶体学检测到。在这里，我们表明，即使在相应晶体结构中不容易观察到溶剂的存在，通过五配位血红素吸收光谱的细微变化，也可以在肌红蛋白突变体的远端腔中检测到水。在野生型（wt）脱氧肌红蛋白的晶体结构中，在远端血红素口袋内可以看到一个定义明确、与远端组氨酸（His64）氢键结合的非配位水分子。这个水分子的位移会降低配体进入wt Mb的速率，并且我们之前已经表明，在MbCO复合物激光光解后，这个水分子的进入很容易通过光学方法检测到。然而，对于L29F和V68L Mb，即使His64仍然存在，并且观察到配体结合速率缓慢表明存在内部水，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。相比之下，激光光解后产生的L29F和V68L脱氧肌红蛋白可见吸收带的时间分辨扰动检测到了这些变体远端口袋内水的进入和显著占有率。因此，五配位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作用提供了一个潜在强大的系统。
