KINETICS OF ENERGY TRANSDUCTION BY BACTERIORHODOPSIN

细菌视紫红质能量转换的动力学

• 批准号: 6432639
• 负责人: richard w hendler
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432639
• 关键词: bacteriorhodopsins; bioenergetics; cellular respiration; chemical kinetics; conformation; cytochrome oxidase; electron transport; enzyme activity; iron oxide; lipopolysaccharides; membrane lipids; oxidation reduction reaction; photosynthesis; viscosity

(A.) In a collaboration with Dr. Joachim Heberle in Germany, we have combined time-resolved optical and FTIR spectroscopies to capitalize on the different abilities of each method to reveal the changes in protein conformation and liganding that accompany spectrally defined transitions during the bacteriorhodopsin (BR) photocycle. The specific goal is to examine native purple membranes (PM) in comparison to Triton-treated and various stages of lipid-reconstituted preparations to look for strong correlations in the kinetics of proton release and uptake with well-defined intermediate steps in the photocycle. All preparations of material are made in our laboratory and the same preparation is examined optically by us and by Heberle in Germany using FTIR. Then both data sets are combined in one large matrix and examined by us using singular value decomposition (SVD). The power of this combined approach was demonstrated by the fact that the optical data alone could be fit to 6 exponentials and the FTIR data alone to 5 exponentials, but the combined data set revealed 7 exponential transitions. This is because there is different information in the two data sets and the combination contains more information than either set alone. The simultaneous SVD deconvolution reveals changes in protein conformation and liganding for each isolated step in the photocycle. The 7-exponential solution split the L->M transition normally seen at 100 us into two steps at 59 and 158 us. It also resolved a step, we originally saw as an M->O step into an M->N and N->O steps. The combined Triton-treated data revealed much slower kinetics and three L- >M transitions, as well as three M->BR transitions, and an absence of the M-fast intermediate. (B.) Our previous work has shown that the neutral lipid, squalene is essential for formation of the M-fast (Mf) intermediate. We find that the neutral solvent, decane, can convert the M-slow (Ms) intermediate into Mf. We now have a variety of ways to alter the relative amounts of Mf and Ms in the BR photocycle: 1. Triton, high pH, high energy actinic light and membrane potential all convert Mf to Ms. 2. Native lipids, particularly squalene with phosphatidyl glycerophosphate (PGP), or decane convert Ms to Mf. The ability to prepare samples with widely different ratios of Mf/Ms should help in elucidating the protein conformational forms that characterize Mf and Ms and perhaps their individual roles in energy transduction. (C.) Use of site-directed mutants to study the importance of a suspected squalene-PGP-acidic aminoacid interaction for Mf activity. Our finding that reconstitution of native photocycle activity by addition of native lipids to Triton-treated PM requires either high salt or pH titration with an apparent pK of ~5, suggests such an interaction. We are using specific mutants where acidic residues exist closely to the lipids of the membrane to test this hypothesis, specifically, D38N, D36N, D102N/D104N, and D36N/D38N/D102N. A common feature is that the loss of D36 and D38 does result in a dramatic decrease in the amount of Mf and in the buildup of a very slow form of M. (D.) A paper was published (JACS, (1999) 121:1385-1386) with Ad Bax showing that magnetically oriented PM membranes can be used to provide the required weak degree macromolecular alignment in strong magnetic fields to allow the NMR measurement of dipolar couplings of certain added soluble proteins. - bionergetics, spectral deconvolution, SVD, time-resolved FTIR and optical spectra

（A.）在与德国的Joachim Heberle博士的合作中，我们将时间分辨的光学和FTIR光谱镜结合在一起，以利用每种方法的不同能力来揭示蛋白质构象的变化以及在细菌hopopsin（BR）光纤维中伴随光谱过渡的蛋白质构象变化。具体目标是与特里顿处理的脂质重新成立的制剂相比，检查天然紫色膜（PM），以在质子释放的动力学和摄取中寻找较强的相关性，并在光周期内具有明确的中间步骤。所有材料的制剂都是在我们的实验室中进行的，我们使用FTIR在德国和德国的Heberle进行了光学检查。然后，两个数据集都在一个大矩阵中组合在一起，并使用单数值分解（SVD）对我们进行了检查。这种组合方法的功能证明了单独的光学数据可以适合6个指数，而仅FTIR数据仅适用于5个指数，但是组合的数据集揭示了7个指数转换。这是因为两个数据集中有不同的信息，并且该组合包含的信息比单独的任何一个集合。同时的SVD反卷积揭示了光循环中每个分离的步骤的蛋白质构象和配体变化。 7个指数解决方案通常在100下将L-> m转变分为59和158 US的两个步骤。它还解决了一个步骤，我们最初将其视为M-> o进入M-> n和n-> o步骤。结合的Triton处理的数据显示，动力学和三个L-> m的转变以及三个M-> BR转变以及M-fast中间体的缺失。 （B.）我们以前的工作表明，中性脂质，沙列烯对于形成M-fast（MF）中间体至关重要。我们发现中性溶剂，脱烷可以将中间体中间的M-Slow（MS）转换为MF。现在，我们有多种方法可以改变BR光循环中MF和MS的相对量：1。Triton，高pH，高能活化光的光和膜电位，所有这些都将MF转换为M. 2. M. 2.天然脂质，特别是与磷脂酰甘油磷酸磷酸盐（PGP）（PGP）（PGP），或将MF转换为MF的天然脂质。制备具有广泛不同比率MF/MS的样品的能力应有助于阐明表征MF和MS的蛋白质构象形式，也许是它们在能量转导中的各个角色。 （C.）使用位置定向的突变体研究可疑的小矛列烯-PGP氨基酸氨基酸相互作用在MF活性中的重要性。我们的发现，通过在Triton处理的PM中添加天然脂质来重建天然光循环活性，需要高盐或pH滴定，明显的PK 〜5，这表明这种相互作用。我们使用的是特定的突变体，其中酸性残基与膜的脂质紧密存在，以检验该假设，特别是D38N，D36N，D102N/D104N和D36N/D38N/D102N。一个共同的特征是，D36和D38的损失确实会导致MF的数量急剧减少，并且在非常缓慢的M.（D.）发表了一份论文（Jacs，（1999）121：1385-1386）中，AD BAX允许使用磁性的PM MEMBRANES，可以允许使用AD BAX，以提供磁性的弱度MACRORECMACRORECTOUNT MACR：测量某些添加的可溶蛋白的偶极耦合。 - 生物测量学，光谱反卷积，SVD，时间分辨FTIR和光谱