Hass Mathias A S, Thuesen Marianne H, Christensen Hans E M, Led Jens J
Department of Chemistry, University of Copenhagen, The H. C. Ørsted Institute, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
J Am Chem Soc. 2004 Jan 28;126(3):753-65. doi: 10.1021/ja030366m.
An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the micros-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena variabilis (A.v. PCu) (Ma, L.; Hass, M. A. S.; Vierick, N.; Kristensen, S. M.; Ulstrup, J.; Led, J. J. Biochemistry 2003, 42, 320-330). The R1 and R2 relaxation rates of the backbone 15N nuclei were measured at a series of pH and temperatures on an 15N labeled sample of A.v. PCu, and the 15N chemical shifts were obtained from a series of HSQC spectra recorded in the pH range from 4 to 8. From the R1 and R2 relaxation rates, the contribution, Rex, to the transverse relaxation caused by the exchanges between the different allo-states of the protein were determined. Specifically, it is demonstrated that accurate Rex terms can be obtained from the R1 and R2 rates alone in the case of relatively rigid proteins with a small rotational anisotropy. The Rex terms belonging to the same exchange process were identified on the basis of their pH dependences. Subsequently the identifications were confirmed quantitatively by the correlation between the Rex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the Rex terms of 15N nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis of the exchange terms shows that the observed micros-ms dynamics in A.v. PCu are caused primarily by the protonation/deprotonation of two histidine residues, His92 and His61, His92 being ligated to the Cu(I) ion. Also the exchange rate of the protonation/deprotonation process of His92 and its pH and temperature dependences were determined, revealing a reaction pathway that is more complex than a simple specific-acid/base catalysis. Finally, the approach allows a differentiation between two-site and multiple-site exchange processes, thus revealing that the protonation/deprotonation of His61 is at least a three-site exchange process. Overall, the approach makes it feasible to obtain exchange rates that are sufficiently accurate and versatile for studies of the kinetics and the mechanisms of local protein dynamics on the sub-millisecond time scale.
本文提出了一种方法，可在微秒至毫秒时间尺度上对蛋白质中的构象交换过程进行详细的定量表征。该方法依赖于对核磁共振弛豫速率和化学位移变化的联合分析，并且要求能够独立于弛豫速率来确定交换物种的化学位移。通过对先前在蓝铜蛋白——来自可变鱼腥藻（A.v. PCu）的质体蓝素还原形式中观察到的构象交换过程进行详细分析，证明了该方法的适用性（Ma, L.; Hass, M. A. S.; Vierick, N.; Kristensen, S. M.; Ulstrup, J.; Led, J. J. Biochemistry 2003, 42, 320 - 330）。在一系列pH值和温度下，对15N标记的A.v. PCu样品测量了主链15N核的R1和R2弛豫速率，并从在4至8的pH范围内记录的一系列HSQC光谱中获得了15N化学位移。根据R1和R2弛豫速率，确定了蛋白质不同变构状态之间的交换对横向弛豫的贡献Rex。具体而言，证明了对于具有小旋转各向异性的相对刚性蛋白质，仅从R1和R2速率就能获得准确的Rex项。根据其pH依赖性识别出属于同一交换过程的Rex项。随后，通过Rex项与交换物种相应化学位移差异之间的相关性对识别结果进行了定量确认。通过这种方法，可将蛋白质中相邻区域的15N核的Rex项分配到同一交换过程。此外，对交换项的分析表明，在A.v. PCu中观察到的微秒至毫秒动力学主要是由两个组氨酸残基His92和His61的质子化/去质子化引起的，His9与Cu(I)离子配位。还确定了His92质子化/去质子化过程的交换速率及其pH和温度依赖性，揭示了一条比简单的特定酸/碱催化更复杂的反应途径。最后，该方法能够区分两点和多点交换过程，从而揭示His61的质子化/去质子化至少是一个三点交换过程。总体而言，该方法使得获得足够准确且通用的交换速率成为可能，以用于研究亚毫秒时间尺度上局部蛋白质动力学的动力学和机制。
