Kent M S, Yim H, Sasaki D Y, Satija S, Majewski J, Gog T
Department 1851, Sandia National Laboratories, Albuquerque, New Mexico 87185, USA.
Langmuir. 2004 Mar 30;20(7):2819-29. doi: 10.1021/la036207y.
The adsorption of myoglobin to Langmuir monolayers of a metal-chelating lipid in crystalline phase was studied using neutron and X-ray reflectivity (NR and XR) and grazing incidence X-ray diffraction (GIXD). In this system, adsorption is due to the interaction between chelated divalent copper or nickel ions and the histidine moieties at the outer surface of the protein. The binding interaction of histidine with the Ni-IDA complex is known to be much weaker than that with Cu-IDA. Adsorption was examined under conditions of constant surface area with an initial pressure of 40 mN/m. After approximately 12 h little further change in reflectivity was detected, although the surface pressure continued to slowly increase. For chelated Cu2+ ions, the adsorbed layer structure in the final state was examined for bulk myoglobin concentrations of 0.10 and 10 microM. For the case of 10 microM, the final layer thickness was approximately 43 A. This corresponds well to the two thicker dimensions of myoglobin in the native state (44 A x 44 A x 25 A) and so is consistent with an end-on orientation for this disk-shaped protein at high packing density. However, the final average volume fraction of amino acid segments in the layer was 0.55, which is substantially greater than the value of 0.44 calculated for a completed monolayer from the crystal structure. This suggests an alternative interpretation based on denaturation. GIXD was used to follow the effect of protein binding on the crystalline packing of the lipids and to check for crystallinity within the layer of adsorbed myoglobin. Despite the strong adsorption of myoglobin, very little change was observed in the structure of the DSIDA film. There was no direct evidence in the XR or GIXD for peptide insertion into the lipid tail region. Also, no evidence for in-plane crystallinity within the adsorbed layer of myoglobin was observed. For 0.1 microM bulk myoglobin concentration, the average segment volume fraction was only 0.13 and the layer thickness was < or = 25 A. Adsorption of myoglobin to DSIDA-loaded with Ni2+ was examined at bulk concentrations of 10 and 50 microM. At 10 microM myoglobin, the adsorbed amount was comparable to that obtained for adsorption to Cu2+-loaded DSIDA monolayers at 0.1 M. But interestingly, the adsorbed layer thickness was 38 A, substantially greater than that obtained at low coverage with Cu-IDA. This indicates that either there are different preferred orientations for isolated myoglobin molecules adsorbed to Cu-IDA and Ni-IDA monolayer films or else myoglobin denatures to a different extent in the two cases. Either interpretation can be explained by the very different binding energies for individual interactions in the two cases. At 50 microM myoglobin, the thickness and segement volume fraction in the adsorbed layer for Ni-IDA were comparable to the values obtained with Cu-IDA at 10 microM myoglobin.
利用中子反射率和X射线反射率（NR和XR）以及掠入射X射线衍射（GIXD）研究了肌红蛋白在晶相金属螯合脂质Langmuir单分子层上的吸附情况。在该体系中，吸附是由于螯合的二价铜或镍离子与蛋白质外表面的组氨酸部分之间的相互作用。已知组氨酸与镍-亚氨基二乙酸（Ni-IDA）络合物的结合相互作用比与铜-亚氨基二乙酸（Cu-IDA）的弱得多。在恒定表面积、初始压力为40 mN/m的条件下检测吸附情况。大约12小时后，尽管表面压力继续缓慢增加，但反射率几乎没有进一步变化。对于螯合的Cu2+离子，在本体肌红蛋白浓度为0.10和10 μM的情况下研究了最终状态下的吸附层结构。对于10 μM的情况，最终层厚度约为43 Å。这与天然状态下肌红蛋白的两个较大尺寸（44 Å×44 Å×25 Å）非常吻合，因此与这种盘状蛋白质在高堆积密度下的端对端取向一致。然而，层中氨基酸片段的最终平均体积分数为0.55，大大高于根据晶体结构计算出的完整单分子层的0.44值。这表明基于变性的另一种解释。GIXD用于跟踪蛋白质结合对脂质晶体堆积的影响，并检查吸附的肌红蛋白层内的结晶度。尽管肌红蛋白有很强的吸附作用，但在DSIDA膜的结构中观察到的变化很小。在XR或GIXD中没有直接证据表明肽插入脂质尾部区域。此外，在肌红蛋白吸附层内也没有观察到面内结晶度的证据。对于本体肌红蛋白浓度为0.1 μM的情况，平均片段体积分数仅为0.13，层厚度≤25 Å。在本体浓度为10和50 μM的情况下研究了肌红蛋白在负载Ni2+的DSIDA上的吸附。在10 μM肌红蛋白时，吸附量与在0.1 M时吸附到负载Cu2+的DSIDA单分子层上的吸附量相当。但有趣的是，吸附层厚度为38 Å，大大大于在低覆盖率下与Cu-IDA获得的厚度。这表明要么吸附到Cu-IDA和Ni-IDA单分子层膜上的孤立肌红蛋白分子有不同的优选取向，要么两种情况下肌红蛋白变性程度不同。两种解释都可以用两种情况下单个相互作用的结合能差异很大来解释。在50 μM肌红蛋白时，Ni-IDA吸附层的厚度和片段体积分数与在10 μM肌红蛋白时用Cu-IDA获得的值相当。
