Hansen D Flemming, Vallurupalli Pramodh, Kay Lewis E
Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2009 Sep 9;131(35):12745-54. doi: 10.1021/ja903897e.
An understanding of many biological processes can only be achieved through studies of the structure (enthalpy) and motions (entropy) of the key molecules that are involved, including those that are formed only transiently and with low population. These transiently formed, low populated states are invisible to most biophysical techniques but in many cases they can be studied in detail using relaxation dispersion NMR spectroscopy. Relaxation dispersion methodology has recently been described for the measurement of protein backbone excited state chemical shifts as well as bond vector orientations, which form the basis for structural studies of these invisible conformers. It is of interest to extend such studies by quantifying motional parameters of the excited state, providing a more complete description of the energy landscape that drives the biochemical event in question. Herein we describe a relaxation dispersion method for measuring site-specific motional parameters of methyl containing residues in the excited state. The approach is applied to the invisible unfolded state of the G48M Fyn SH3 domain that is in exchange with the folded conformation. Not surprisingly, the degree of disorder is in general higher in the unfolded state than in the folded conformer, although there is some ordering of side-chains in the unfolded state toward the C-terminal region of the domain. The development of the present methodology provides the first step toward characterizing the motional properties of invisible conformers, complementing the structural information that is already available from relaxation dispersion studies.
只有通过研究涉及的关键分子的结构（焓）和运动（熵），包括那些仅短暂形成且丰度较低的分子，才能理解许多生物过程。这些短暂形成、丰度较低的状态对大多数生物物理技术来说是不可见的，但在许多情况下，可以使用弛豫色散核磁共振光谱对其进行详细研究。最近已经描述了弛豫色散方法，用于测量蛋白质主链激发态化学位移以及键向量方向，这些构成了对这些不可见构象体进行结构研究的基础。通过量化激发态的运动参数来扩展此类研究是很有意义的，这可以更完整地描述驱动相关生化事件的能量图景。在此，我们描述了一种弛豫色散方法，用于测量激发态中含甲基残基的位点特异性运动参数。该方法应用于与折叠构象发生交换的G48M Fyn SH3结构域的不可见未折叠状态。不出所料，未折叠状态下的无序程度通常高于折叠构象体，尽管在未折叠状态下，结构域C端区域的侧链存在一些有序排列。本方法的开发为表征不可见构象体的运动特性迈出了第一步，补充了弛豫色散研究中已有的结构信息。
