Nature has evolved sophisticated strategies for engineering hard tissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. The remarkable material properties of bone and teeth thus result from the activities of proteins that function at the organic-inorganic interface. The underlying molecular mechanisms that control biomineralization are of significant interest to both medicine and dentistry, as disruption of biomineralization processes can lead to bone and tooth demineralization, atherosclerotic plaque formation, artificial heart valve calcification, kidney and gall stone build-up, dental calculus formation, and arthritis [1–3]. A better understanding of the biomolecular mechanisms used to promote or retard crystal growth could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. Similarly, a better understanding of how these proteins recognize and assemble in bioactive form on inorganic mineral phases could also aid in the development of surface coatings to improve the biocompatibility of implantable biomaterials and for hard tissue engineering and regeneration technologies. At the level of fundamental science, it is important to note the lack of molecular structure information available for biomineralization proteins in general, and in particular for mammalian proteins that directly control calcification processes in hard tissue. Even the most fundamental questions about how the proteins interact at the biomineral surface, such as their general structure and orientation on the calcium phosphate surfaces, or whether the acidic residues are truly interacting directly with the crystal surface, remain largely uncharacterized at the experimental level. In order to develop a better structure-function level understanding of protein-crystal molecular recognition, we have begun to utilize solid-state NMR techniques to determine the molecular structure of proteins and peptides on calcium phosphate surfaces. In addition, these same techniques have provided interesting molecular dynamics information for the proteins on the biomineral surface. In this review, we will highlight recent work that is providing insight into the structure and crystal recognition mechanisms of an exemplary salivary protein model system, but which also provides a general approach to studying protein-crystal interactions in molecular detail. Understanding the function of a biomineralization protein requires that the secondary and tertiary structure of the molecule be defined within its biological context, i.e. the protein in contact with the crystal surface. In addition, the precise nature of the interactions between the protein and the crystal which underlie the recognition process must be understood. This requires knowledge of the contacts formed between the amino acid side chains of the protein and the ions in the crystal faces. The involvement of water molecules in these interactions must be understood as well. Current investigations of protein-mineral interactions are frequently conducted with techniques that characterize the macroscopic behavior of proteins in the presence of mineral crystals. Equilibrium properties such as protein-crystal binding constants are derived via adsorption isotherm measurements, where data are usually analyzed by assuming a simple Langmuir model of protein adsorption onto the crystal faces. But the most commonly-used approach for determining protein-crystal interactions in vitro are kinetic experiments in which a small amount of protein is dissolved in a saturated solution of a particular inorganic salt and the time required for crystals to form is compared to a control solution in which no protein is present. Assays also exist for determining selective binding of a particular crystal face by a protein as well as oriented nucleation of crystals in the presence of acidic proteins [4]. Recently, isothermal titration calorimetry has been used to determine binding enthalpies and binding affinities for proteins to mineral surfaces [5]. However, to extend beyond macroscopic aspects of protein-crystal interactions, high resolution spectroscopic methods must be used to provide information about the atomic level structure of the protein on the crystal face, under physical conditions that are biologically relevant (physiological levels of hydration and pH). Information about the secondary structural motifs and tertiary folding that characterize the adsorbed protein, together with information on the exposure of protein side chains to the crystal face, may lead to an understanding of how particular proteins promote or inhibit nucleation. The lack of high resolution structural data for proteins on surfaces is the result of a lack of high resolution structural methods that can be brought to bear on relevant problems. The conventional methods of high resolution structural biology, i.e. X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, have provided information on a few biomineralization proteins in the pure crystalline and solution states [6–9], but both techniques are severely limited in their abilities to elucidate the structures of proteins on biomineral surfaces. Although traditional surface science methods like photo-electron spectroscopy and NEXAFS have provided important information on protein adsorbed onto planar surfaces, and in particular may be used to characterize the degree of long range ordering in systems of adsorbed proteins on polymer surfaces as well as average structural properties, these techniques have yet to provide detailed atomic-level structural information for surface-adsorbed proteins. In addition, surface diffraction methods and many optical techniques are not applicable to proteins adsorbed onto surfaces of porous materials (e.g. porous plastics) or to other surfaces lacking long-range ordering. To fully appreciate the utility of solid state NMR in the study of protein structure at biomaterial interfaces, it is important to recognize the complex nature of the protein–surface problem. There is first the familiar structural aspect, alluded to briefly above, which includes defining the secondary and tertiary structures of the adsorbed protein and by implication any structural changes which occur upon binding to the surface. Secondly, the structure and chemical composition of the crystal surface in contact with the protein side chains must also be understood. The dynamics of the adsorbed protein are a less familiar but no less important aspect. It is desirable that the protein be observed on the surface under biologically relevant conditions, i.e. fully hydrated. Dehydration of the sample may alter not only the structure of the protein from its biologically relevant form but may quench the dynamics of the protein on the surface. Here we refer to both whole-molecule dynamics describing the protein’s rigid body kinematics on the surface and to internal dynamics wherein the protein’s conformation may be labile on the NMR time scale.

中文翻译：

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蛋白质-矿物质界面分子识别的固态核磁共振研究

大自然已经进化出复杂的策略，通过蛋白质和最终细胞与无机矿物相的相互作用来设计硬组织。因此，骨骼和牙齿卓越的材料特性是由在有机-无机界面发挥作用的蛋白质的活性造成的。控制生物矿化的潜在分子机制对医学和牙科都具有重要意义，因为生物矿化过程的破坏可能导致骨骼和牙齿脱矿、动脉粥样硬化斑块形成、人造心脏瓣膜钙化、肾脏和胆结石积聚、牙结石形成和关节炎[1-3]。更好地了解用于促进或延迟晶体生长的生物分子机制可以为骨科、心脏病学、泌尿学和牙科中钙化抑制剂和促进剂的开发提供重要的设计原则。同样，更好地了解这些蛋白质如何识别并以生物活性形式在无机矿物相上组装也有助于开发表面涂层，以提高可植入生物材料的生物相容性以及硬组织工程和再生技术。在基础科学层面，重要的是要注意一般生物矿化蛋白，特别是直接控制硬组织钙化过程的哺乳动物蛋白，缺乏可用的分子结构信息。即使是关于蛋白质如何在生物矿物表面相互作用的最基本问题，例如它们在磷酸钙表面上的一般结构和方向，或者酸性残基是否真正与晶体表面直接相互作用，在实验水平上仍然很大程度上未被表征。为了更好地理解蛋白质晶体分子识别的结构-功能水平，我们已经开始利用固态核磁共振技术来确定磷酸钙表面上蛋白质和肽的分子结构。此外，这些相同的技术还为生物矿物表面的蛋白质提供了有趣的分子动力学信息。在这篇综述中，我们将重点介绍最近的工作，这些工作不仅深入了解了示例性唾液蛋白模型系统的结构和晶体识别机制，而且还提供了研究分子细节中蛋白质-晶体相互作用的通用方法。了解生物矿化蛋白的功能需要在其生物学背景下定义分子的二级和三级结构，即与晶体表面接触的蛋白质。此外，必须了解识别过程中蛋白质和晶体之间相互作用的精确性质。这需要了解蛋白质的氨基酸侧链与晶面中的离子之间形成的接触。还必须了解水分子在这些相互作用中的参与。目前对蛋白质-矿物质相互作用的研究经常使用表征蛋白质在矿物晶体存在下的宏观行为的技术。蛋白质-晶体结合常数等平衡特性是通过吸附等温线测量得出的，通常通过假设蛋白质吸附到晶体面上的简单朗缪尔模型来分析数据。但体外确定蛋白质-晶体相互作用的最常用方法是动力学实验，其中将少量蛋白质溶解在特定无机盐的饱和溶液中，并将晶体形成所需的时间与对照溶液进行比较其中不存在蛋白质。还存在用于确定蛋白质对特定晶面的选择性结合以及在酸性蛋白质存在下晶体定向成核的测定方法[4]。最近，等温滴定量热法已被用来确定蛋白质与矿物表面的结合焓和结合亲和力[5]。然而，为了超越蛋白质-晶体相互作用的宏观方面，必须使用高分辨率光谱方法在生物学相关的物理条件（水合的生理水平和 pH 值）下提供有关晶体表面上蛋白质的原子级结构的信息。 ）。有关表征吸附蛋白质的二级结构基序和三级折叠的信息，以及有关蛋白质侧链暴露于晶面的信息，可能有助于了解特定蛋白质如何促进或抑制成核。表面蛋白质高分辨率结构数据的缺乏是由于缺乏可用于解决相关问题的高分辨率结构方法的结果。高分辨率结构生物学的传统方法，即X射线晶体学和溶液核磁共振（NMR）波谱，已经提供了一些纯晶体和溶液状态的生物矿化蛋白质的信息[6-9]，但这两种技术都严重依赖他们阐明生物矿物表面蛋白质结构的能力有限。尽管光电子能谱和 NEXAFS 等传统表面科学方法提供了有关平面表面吸附蛋白质的重要​​信息，特别是可用于表征聚合物表面吸附蛋白质系统的长程有序程度以及平均结构尽管这些技术尚未提供表面吸附蛋白质的详细原子级结构信息。此外，表面衍射方法和许多光学技术不适用于吸附在多孔材料表面的蛋白质（例如 多孔塑料）或其他缺乏远程有序的表面。为了充分认识固态核磁共振在生物材料界面蛋白质结构研究中的效用，重要的是要认识到蛋白质表面问题的复杂性。首先是上面简要提到的熟悉的结构方面，其中包括定义吸附蛋白质的二级和三级结构，并暗示在与表面结合时发生的任何结构变化。其次，还必须了解与蛋白质侧链接触的晶体表面的结构和化学成分。吸附蛋白质的动力学是一个不太熟悉但同样重要的方面。理想的是在生物学相关条件下，即完全水合的情况下，在表面上观察到蛋白质。样品脱水不仅可能改变蛋白质的生物学相关形式的结构，还可能淬灭表面蛋白质的动力学。在这里，我们既指描述蛋白质表面刚体运动学的全分子动力学，也指内部动力学，其中蛋白质的构象在 NMR 时间尺度上可能不稳定。