Energetics Of The Interaction Between Water & Membranes

这项工作有两个方面。首先是确定溶液中分子和离子之间形成非共价键所需的能量。第二个是了解形成肽离子的能量与其碎片化的类型和程度之间的关系。 非共价键的能量学知识，特别是那些涉及水和生物重要分子的能量学知识，对于理解分子相互作用和与它们不可或缺的构象变化至关重要。本研究涉及使用平衡离子-分子反应化学方法确定热力学量Δ H（std）、Δ S（std）和Δ G（std）298。水合热力学值由在0-136摄氏度的温度范围内在离子源中的水分压在0和100毫托之间的范围内测量的平衡常数计算。对至少4种水合状态进行平衡离子强度测量，零至3个水分子与核心离子相关，并包括至少60种水分压和温度的组合，涵盖了实验变量的范围。结果已经获得了三种不同类型的分子，最近集中在氨基酸。在过去的一年里，我们已经完成了对所有20种必需氨基酸的测量，这些氨基酸中的每一种都含有多达3个水分子。这些广泛测量的最引人注目的结果是，与先前研究的其他分子或原子离子不同，与连续加入沃茨到任何氨基酸中相关的焓变化被发现几乎是相等的。这样做的结果是，连续的沃茨的熵变，不像所有以前的工作，依赖于氨基酸离子的水合状态。我们解释这些结果意味着氨基酸水合作用在能量上非常相似，但水分子所在的位点强烈地受离子的两性离子性质的影响。我们还发现，形成第一水合物种的自由能与氨基酸疏水性的公布值显着相关。 在离子能量学的第二个领域，我们已经开始研究激光能量密度和肽离子断裂之间的关系。这种类型的研究是优化MALDI TOF/TOF实验的基础，用于肽测序。在这些研究中，我们获得肽片段化光谱，通常5000激光射击，在单分子分解和碰撞诱导解离（CID）模式。我们能够容易地跟踪所考虑的每种肽的两个时间点，即，源内碎裂由激光发射后1微秒内形成的离子组成，而更长的、依赖于质量的碎裂发生在仪器的碰撞室内。到目前为止，我们已经研究了两个模型肽，亮氨酸脑啡肽，YGGFL，（LeuEnk）和des-Arg-1缓激肽，PPGFSPFR，（desRB）在整个范围内的激光能量密度。我们观察到LeuEnk在比desRB低得多的能量密度下表现出碎片化的开始。然而，更重要的是，我们已经能够证明，作为增加激光能量密度的函数的碎片形成作为一组连续的而不是竞争的反应进行。这对于肽测序是非常重要的，因为为了从一系列连续反应中获得完整的碎片离子集，必须形成具有足够内能的感兴趣的肽离子以支持整个分解链。在肽混合物的情况下，即，在测序肽时的真实的情况下，只有在MADI实验中才能提供必要量的内能。我们同时开发的动力学模型，这些分解使用赖斯-拉姆斯伯格-卡塞尔-马库斯（RRKM）的形式主义气相动力学。除了对碎片化建模之外，计算还将定义肽离子温度的下限。有了这些信息，我们将能够第一次估计激光能量传递到气相离子的分数。此外，我们将有一种手段，而不是纯粹的结晶，选择和优化MALDI矩阵和激光频率。

There are two aspects to this work. The first is the determination of the energies required for the formation of non-covalent bonds between molecules and ions in solution. The second is to understand the relationship between the energy applied in forming a peptide ion and the type and extent of its fragmentation. Knowledge of the energetics of non-covalent bonds, particularly those involving water and biologically significant molecules, is fundamental to understanding molecular interactions and the changes in conformations that are integral to them. This research involves determining the thermodynamic quantities deltaH(std), deltaS(std), and deltaG(std)298 using the approach of equilibrium ion-molecule reaction chemistry. Hydration thermodynamics values are calculated from equilibrium constants measured over a temperature range of 0-136 degrees C at water partial pressures in the ion source ranging between zero and 100 mtorr. Equilibrium ion intensity measurements were made for at least 4 hydration states, i.e., zero through 3 water molecules associated with a core ion, and include at least 60 combinations of water partial pressures and temperatures covering the range of experimental variables. Results have been obtained for three different classes of molecules, most recently focusing on amino acids. In the past year we have completed measurements on all twenty of the essential amino acids for clusters containing up to three water molecules each. The most striking result of these extensive measurements is that, unlike other molecular or atomic ions studied previously, the enthalpy changes associated with the successive addition of waters to any of the amino acids were found to be virtually equivalent. The consequence of this is that the entropy changes for the successive waters are, unlike all previous work, dependent on the hydration state of the amino acid ion. We interpret these results to mean that amino acid hydrations are energetically very similar, but that the sites at which the water molecules are located are strongly directed by the zwitterionic nature of the ions. We have also found that the free energy for the formation of the first hydrated species is correlated significantly with the published values for amino acid hydrophobicity. In a second area of ion energetics, we have begun investigating the relationship between laser fluence and peptide ion fragmentation. This type of study is fundamental to optimizing MALDI TOF/TOF experiments for the purpose of peptide sequencing. In these studies we obtain peptide fragmentation spectra, typically 5000 laser shots, in both the unimolecular decomposition and collision induced dissociation (CID) modes. We have the ability to easily follow two time points for each peptide considered, i.e., the in-source fragmentation consisting of ions formed within 1 usec after the laser firing and the longer, mass dependent fragmentation occurring within the instrument's collision cell. To date we have studied the fragmentation of two model peptides, leucine enkephalin, YGGFL, (LeuEnk) and des-Arg-1 bradykinin, PPGFSPFR, (desRB) over the full range of laser fluence available. We observe that LeuEnk exhibits an onset of fragmentation at a much lower fluence than desRB. More importantly, however, we have been able to demonstrate that fragment formation as a function of increasing laser fluence proceeds as a set of consecutive rather than competing reactions. The implications of this are very significant for peptide sequencing since, in order to obtain a full set of fragment ions from a series of consecutive reactions, it is necessary that the peptide ion of interest be formed with sufficient internal energy to support the entire chain of decompositions. In the context of a peptide mixture, i.e., the real situation when sequencing a peptide, the necessary amount of internal energy can be supplied only in a MADI experiment. We are simultaneously developing a kinetic model for these decompositions using the Rice-Ramsberger-Kassel-Marcus (RRKM) formalism for gas phase kinetics. In addition to modeling fragmentation, the calculations will define a lower limit of the peptide ion temperatures. With this information, we will be able for the first time to estimate the fraction of laser energy delivered to gas phase ions. Furthermore, we will have a means, other than pure empiricism, to select and optimize both MALDI matrix and laser frequency.