STRUCTURAL DETERMINATION OF PARAMAGNETIC CENTER IN PUTIDAREDOXIN

腐胺氧还蛋白顺磁中心的结构测定

• 批准号: 6355159
• 负责人: MARINA L BENNATI
• 金额: $ 2.03万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355159
• 关键词: biological products; biomedical resource; proteins

Structural determination of paramagnetic proteins by solid state and solution NMR techniques is limited by severe line broadening due to the strong hyperfine interaction between the nuclei and the paramagnetic center. This prevents the use of standard NMR structural methods within a few angstroms of the metal center. In this context, high frequency (140 GHz) pulsed ENDOR techniques are ideally suited to extract hyperfine interactions in the vicinity of the paramagnetic center. We have been investigating a 2-Fe 2-S ferredoxin putidaredoxin (Pdx), where the metal center displays an effective spin S = 1/2 in the reduced form (Fe" - Fe") and the cluster is ligated by four cysteinyl sulfurs. We were able to record 140 GHz spin-echo detected spectra at low temperatures (10 K) with excellent signal-to-noise (> 50) and determine the g-anisotropy to g,, = 2.02 and g_L = 1.94. TI-relaxation of the Fe-cluster was measured between 9 and 40 K. We found short T,- values of 50 gs at 9 K and a more complex biexponential behavior between 15 - 40 K, likely due to the thermal population of higher excited spin states. A special ENDOR resonator for efficient irradiation of low-gamma nuclei was developed and preliminary "C-Mims-ENDOR spectra were recorded at 10 K. In contrast to ENDOR data previously reported in the literature at low EPR frequencies (35 GHz), the ' 3C larmor frequency at 5 Tesla (53 MHz) is well separated from other nuclei and we observe symmetric ENDOR lineshapes around the larmor frequency. In a "C uniformly labeled protein, two distinct hyperfine splitting have been observed, which we attribute to the cystein methylene groups. Further experiments with selectively labeled proteins are in progress and will permit unambiguous assignments of the observed hyperfine couplings.

顺磁性蛋白质的固态结构测定 溶液核磁共振技术受到严重谱线展宽的限制 原子核与原子核之间的强超精细相互作用 顺磁中心。 这妨碍了标准 NMR 结构的使用 金属中心几埃以内的方法。 在此背景下， 高频 (140 GHz) 脉冲 ENDOR 技术非常适合 提取顺磁附近的超精细相互作用 中心。 我们一直在研究 2-Fe 2-S 铁氧还蛋白 恶臭氧还蛋白 (Pdx)，其中金属中心显示有效自旋 S = 1/2 的还原形式 (Fe" - Fe") 并且簇通过以下方式连接 四个半胱氨酰硫。 我们能够记录 140 GHz 自旋回波 在低温 (10 K) 下检测到的光谱具有出色的 信噪比 (> 50) 并确定 g 各向异性为 g,, = 2.02 g_L = 1.94。 Fe 团簇的 TI 弛豫在 9 和 40 K。我们发现在 9 K 及更高温度下 T,- 值为 50 gs 15 - 40 K 之间的复杂双指数行为，可能是由于 较高激发自旋态的热布居。 特别的ENDOR 开发出用于低伽马核有效照射的谐振器 初步的“C-Mims-ENDOR 光谱是在 10 K 下记录的。 与之前文献报道的 ENDOR 数据相比，低 EPR 频率 (35 GHz)，5 特斯拉时的 3C 拉莫尔频率 (53 MHz）与其他原子核很好地分离，我们观察到对称 ENDOR 围绕拉莫尔频率的线形。 在“C统一 标记的蛋白质，观察到两种不同的超精细分裂， 我们将其归因于半胱氨酸亚甲基。 更远 选择性标记蛋白质的实验正在进行中，并将 允许对观察到的超精细耦合进行明确的分配。