Yang Q X, Huang F Y, Lin T H, Gelbaum L, Howell E E, Huang T H
Division of Structural Biology, Academia Sinica, Nankang, Taipei, Taiwan.
Solid State Nucl Magn Reson. 1996 Dec;7(3):193-201. doi: 10.1016/0926-2040(95)01223-0.
We have employed deuterium NMR techniques to determine the dynamics of trimethoprim (TMP) in a binary complex with dihydrofolate reductase (DHFR) or in a ternary complex with DHFR and cofactor NADP+ in the fully hydrated state. TMP was deuterated at the following positions: (2',6'-D2)TMP, (3'-Ome-D3)TMP and (3',4'-Ome-D6)TMP. Dynamics of TMP were deduced from lineshape simulation and relaxation measurements of the deuterium NMR powder spectra of the three samples obtained at various temperatures. The results showed that in the polycrystalline state the TMP molecule is very rigid. The only detectable motion is the methyl group rotation at a rate of 10(10) s-1 at 25 degrees C, as determined from simulation of the partially relaxed powder patterns. When bound to DHFR a residual deuterium quadrupole splitting of 140 kHz was observed for (2',6'-D2)TMP at temperatures up to 30 degrees C, suggesting that the benzyl ring in the bound state is also very rigid. In contrast, in the binary complex with DHFR the methoxyl groups of TMP undergo librational motion of 10(7) s-1 about the C3-O bond at an amplitude of 54 degrees for the meta methoxyl group and about the C4-O bond at an amplitude of 70 degrees and similar rate for the para methoxyl group at 30 degrees C. The presence of the cofactor, NADP+, appears to tighten up the binding pocket such that the motion freedom of TMP is more restricted. The rigidity of TMP in a protein complex as revealed by our deuterium NMR results is in accord with the tight binding of TMP to DHFR.
我们采用氘核磁共振技术来确定甲氧苄啶（TMP）与二氢叶酸还原酶（DHFR）形成的二元复合物中，或与DHFR和辅因子NADP⁺在完全水合状态下形成的三元复合物中的动力学情况。TMP在以下位置进行了氘代：（2'，6'-D₂）TMP、（3'-Ome-D₃）TMP和（3'，4'-Ome-D₆）TMP。通过对在不同温度下获得的三个样品的氘核磁共振粉末谱进行线形模拟和弛豫测量，推断出TMP的动力学情况。结果表明，在多晶状态下，TMP分子非常刚性。唯一可检测到的运动是甲基的旋转，在25℃下其速率为10¹⁰ s⁻¹，这是通过对部分弛豫的粉末图谱进行模拟确定的。当与DHFR结合时，在高达30℃的温度下，观察到（2'，6'-D₂）TMP的残余氘四极分裂为140 kHz，这表明结合状态下的苄基环也非常刚性。相比之下，在与DHFR形成的二元复合物中，TMP的甲氧基在30℃下围绕C3 - O键以10⁷ s⁻¹的速率进行摆动运动，间位甲氧基的摆动幅度为54°，围绕C4 - O键摆动，对位甲氧基摆动幅度为70°且速率相似。辅因子NADP⁺的存在似乎使结合口袋收紧，从而使TMP的运动自由度受到更大限制。我们的氘核磁共振结果所揭示的TMP在蛋白质复合物中的刚性与TMP与DHFR的紧密结合相一致。
