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Structure and function of proteins investigated by crystallographic and spectroscopic time-resolved methods.

機譯：通過晶體學和光譜學時間分辨方法研究蛋白質(zhì)的結(jié)構(gòu)和功能。

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Biomolecules play an essential role in performing the necessary functions for life. The goal of this thesis is to contribute to an understanding of how biological systems work on the molecular level. We used two biological systems, beef liver catalase (BLC) and photoactive yellow protein (PYP). BLC is a metalloprotein that protects living cells from the harmful effects of reactive oxygen species by converting H2O2 into water and oxygen. By binding nitric oxide (NO) to the catalase, a complex was generated that mimics the Cat-H2O2 adduct, a crucial intermediate in the reaction promoted by the catalase. The Cat-NO complex is obtained by using a convenient NO generator (1-(N,N-diethylamino)diazen-1-ium-1,2-diolate). Concentrations up to 100∼200 mM are reached by using a specially designed glass cavity. With this glass apparatus and DEANO, sufficient NO occupation is achieved and structure determination of the catalase with NO bound to the heme iron becomes possible. Structural changes upon NO binding are minute. NO has a slightly bent geometry with respect to the heme normal, which results in a substantial overlap of the NO orbitals with the iron-porphyrin molecular orbitals. From the structure of the iron-NO complex, conclusions on the electronic properties of the heme iron can be drawn that ultimately lead to an insight into the catalytic properties of this enzyme.;Enzyme kinetics is affected by additional parameters such as temperature and pH. Additionally, in crystallography, the absorbed X-ray dose may impair protein function. To address the effect of these parameters, we performed time-resolved crystallographic experiments on a model system, PYP. By collecting multiple time-series on PYP at increasing X-ray dose levels, we determined a kinetic dose limit up to which kinetically meaningful X-ray data sets can be collected. From this, we conclude that comprehensive time-series spanning up to 12 orders of magnitude in time can be collected from a single PYP crystal. Time-resolved X-ray data collected at pH's of 4, 7 and 9 demonstrate that pH alters the kinetics of the PYP photocycle dramatically. At pH 4 the photocycle lasts almost one order of magnitude longer in time compared to pH 7. The final intermediate that accumulates at both pH 7 and pH 4 is absent at pH 9. Results from the dose- and the pH-dependent time-resolved crystallographic experiments show that it is imperative to carefully control the conditions under which time-resolved data are collected. With these considerations we collected a comprehensive time-series from nanoseconds to seconds at 14 different temperature settings from -40 °C to 70 °C. Results from time-resolved crystallography are corroborated by employing time-resolved absorption spectroscopy. For this, absorption spectra on crystals and solution are collected by a fast micro-spectrophotometer custom-designed in our lab. We identify kinetic phases of the PYP photocycle at all 14 temperature settings. Relaxation times associated with these phases are temperature-dependent and can be fit by the Van't Hoff-Arrhenius equation. Kinetic modeling yields entropy and enthalpy values at the barriers of the activation solely from the time-resolved crystallographic data. With this, we advance crystallography to a new frontier: the determination of free energy surfaces.;Investigating enzymatic reactions can be challenging, because they are non-cyclic. After one turnover product must be washed away and substrate must be reloaded. A promising approach for routine application can be envisioned at the new 4th generation X-ray sources, such as X-ray free electron lasers (XFELs). With our results we set the scene to comprehensively investigate all kinds of enzymatic reactions with these instruments.
機譯：生物分子在履行生命所必需的功能中起著至關(guān)重要的作用。本文的目的是有助于理解生物系統(tǒng)如何在分子水平上工作。我們使用了兩種生物系統(tǒng)，牛肉肝過氧化氫酶（BLC）和光敏黃色蛋白（PYP）。 BLC是一種金屬蛋白，通過將H2O2轉(zhuǎn)化為水和氧氣來保護活細胞免受活性氧的有害影響。通過將一氧化氮（NO）與過氧化氫酶結(jié)合，生成了模仿Cat-H2O2加合物的復合物，Cat-H2O2加合物是由過氧化氫酶促進的反應(yīng)中的關(guān)鍵中間體。通過使用方便的NO生成器（1-（N，N-二乙氨基）重氮-1-1,2-二醇酸酯）獲得Cat-NO配合物。通過使用專門設(shè)計的玻璃腔，濃度可達到100-200 mM。利用該玻璃設(shè)備和DEANO，可以實現(xiàn)足夠的NO占用，并且可以確定NO與血紅素鐵結(jié)合的過氧化氫酶的結(jié)構(gòu)。 NO結(jié)合后的結(jié)構(gòu)變化很小。 NO相對于血紅素法線具有略微彎曲的幾何形狀，這導致NO軌道與鐵卟啉分子軌道基本重疊。從鐵-一氧化氮配合物的結(jié)構(gòu)中，可以得出血紅素鐵的電子性質(zhì)的結(jié)論，最終可以深入了解該酶的催化性能。酶動力學受溫度和pH等其他參數(shù)的影響。此外，在晶體學中，吸收的X射線劑量可能會損害蛋白質(zhì)功能。為了解決這些參數(shù)的影響，我們在模型系統(tǒng)PYP上進行了時間分辨晶體學實驗。通過以遞增的X射線劑量水平收集PYP上的多個時間序列，我們確定了一個動態(tài)劑量極限，可以收集到該劑量的動力學有意義的X射線數(shù)據(jù)集。據(jù)此，我們得出結(jié)論，可以從單個PYP晶體中收集跨越12個數(shù)量級的時間的綜合時間序列。在4、7和9的pH值下收集的時間分辨X射線數(shù)據(jù)表明，pH值極大地改變了PYP光循環(huán)的動力學。與pH 7相比，在pH 4時，光周期的持續(xù)時間長了幾乎一個數(shù)量級。在pH 9時，最終的中間體在pH 7和pH 4時均不存在。這是由劑量和pH依賴性時間決定的晶體學實驗表明，必須嚴格控制收集時間分辨數(shù)據(jù)的條件?？紤]到這些因素，我們在-40°C至70°C的14種不同溫度設(shè)置下收集了從納秒到秒的全面時間序列。時間分辨晶體學的結(jié)果通過使用時間分辨吸收光譜法得到證實。為此，通過我們實驗室定制的快速微分光光度計收集晶體和溶液的吸收光譜。我們確定在所有14個溫度設(shè)置下PYP光循環(huán)的動力學相。與這些相相關(guān)的弛豫時間取決于溫度，可以通過Van't Hoff-Arrhenius方程擬合。動力學建模僅根據(jù)時間分辨的晶體學數(shù)據(jù)在活化的壁壘處產(chǎn)生熵和焓值。因此，我們將晶體學推向了一個新的領(lǐng)域：自由能表面的確定。研究酶促反應(yīng)可能是具有挑戰(zhàn)性的，因為它們是非環(huán)狀的。一次周轉(zhuǎn)后，必須將產(chǎn)品沖洗掉，并必須重新裝入基材。可以在新的第四代X射線源（例如X射線自由電子激光器（XFEL））上設(shè)想有希望的常規(guī)應(yīng)用方法。憑借我們的結(jié)果，我們?yōu)槔眠@些儀器全面研究各種酶促反應(yīng)奠定了基礎(chǔ)。

著錄項

  • 作者

    Purwar, Namrta.;

  • 作者單位

    The University of Wisconsin - Milwaukee.;

  • 授予單位 The University of Wisconsin - Milwaukee.;
  • 學科 Molecular physics.;Biochemistry.;Biomechanics.
  • 學位 Ph.D.
  • 年度 2013
  • 頁碼 177 p.
  • 總頁數(shù) 177
  • 原文格式 PDF
  • 正文語種 eng
  • 中圖分類
  • 關(guān)鍵詞

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