Biophysics, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
J Phys Chem B. 2012 Sep 27;116(38):11656-61. doi: 10.1021/jp307935g. Epub 2012 Sep 19.
The hierarchical heterogeneous architecture of bone imposes significant challenges to structural and dynamic studies conducted by traditional biophysical techniques. High-resolution solid-state nuclear magnetic resonance (SSNMR) spectroscopy is capable of providing detailed atomic-level structural insights into such traditionally challenging materials. However, the relatively long data-collection time necessary to achieve a reliable signal-to-noise ratio (S/N) remains a major limitation for the widespread application of SSNMR on bone and related biomaterials. In this study, we attempt to overcome this limitation by employing the paramagnetic relaxation properties of copper(II) ions to shorten the (1)H intrinsic spin-lattice (T(1)) relaxation times measured in natural-abundance (13)C cross-polarization (CP) magic-angle-spinning (MAS) NMR experiments on bone tissues for the purpose of accelerating the data acquisition time in SSNMR. To this end, high-resolution solid-state (13)C CPMAS experiments were conducted on type I collagen (bovine tendon), bovine cortical bone, and demineralized bovine cortical bone, each in powdered form, to measure the (1)H T(1) values in the absence and in the presence of 30 mM Cu(II)(NH(4))(2)EDTA. Our results show that the (1)H T(1) values were successfully reduced by a factor of 2.2, 2.9, and 3.2 for bovine cortical bone, type I collagen, and demineralized bone, respectively, without reducing the spectral resolution and thus enabling faster data acquisition. In addition, paramagnetic quenching of particular (13)C NMR resonances on exposure to Cu(2+) ions in the absence of mineral was also observed, potentially suggesting the relative proximity of three main amino acids in the protein backbone (glycine, proline, and alanine) to the bone mineral surface.
骨的层次异质结构对传统生物物理技术进行的结构和动力学研究提出了重大挑战。高分辨率固态核磁共振（SSNMR）光谱技术能够提供对传统上具有挑战性的材料进行详细原子级结构分析的能力。然而，为了在骨和相关生物材料上广泛应用 SSNMR，实现可靠的信噪比（S/N）所需的相对较长的数据采集时间仍然是一个主要限制。在这项研究中，我们试图通过利用铜（II）离子的顺磁弛豫特性来缩短天然丰度（13）C 交叉极化（CP）魔角旋转（MAS）NMR 实验中测量的骨组织的（1）H 固有自旋晶格（T（1））弛豫时间，从而达到加速 SSNMR 数据采集时间的目的。为此，对 I 型胶原蛋白（牛腱）、牛皮质骨和脱矿牛皮质骨进行了高分辨率固态（13）CCPMAS 实验，每种粉末形式均在不存在和存在 30mM Cu（II）（NH（4））（2）EDTA 的情况下测量（1）H T（1）值。我们的结果表明，牛皮质骨、I 型胶原蛋白和脱矿骨的（1）H T（1）值分别成功降低了 2.2、2.9 和 3.2 倍，而不会降低光谱分辨率，从而实现了更快的数据采集。此外，在没有矿物质的情况下暴露于 Cu（2+）离子时，还观察到特定（13）C NMR 共振的顺磁猝灭，这可能表明蛋白质骨架中三个主要氨基酸（甘氨酸、脯氨酸和丙氨酸）与骨矿物质表面的相对接近程度。
