Energetics Of The Interaction Between Water & Membranes

这项工作有两个方面。第一个是测定在溶液中分子和离子之间形成非共价键所需的能量。第二是了解形成多肽离子的能量与其碎裂的类型和程度之间的关系。 了解非共价键的能量学知识，特别是那些涉及水和具有生物意义的分子的知识，是理解分子相互作用和构成分子相互作用的构象变化的基础。本研究涉及用平衡离子-分子反应化学的方法确定热力学参数deltaH(Std)、deltaS(Std)和deltaG(Std)298。水合热力学数值是根据在0-136摄氏度的温度范围内测量的平衡常数计算的，离子源中的水分压在0-100mtorr之间。对至少4种水化状态，即与核心离子有关的0到3个水分子进行了平衡离子强度测量，并包括覆盖实验变量范围的至少60种水分压和温度组合。针对三种不同类型的分子已经获得了结果，最近的结果集中在氨基酸上。在过去的一年里，我们已经完成了对每个包含多达三个水分子的簇的所有20种必需氨基酸的测量。这些广泛的测量最引人注目的结果是，与以前研究的其他分子或原子离子不同，发现与向任何氨基酸连续添加水相关的热变实际上是相等的。这样做的结果是，与以前的所有工作不同，连续水的熵变化取决于氨基酸离子的水合状态。我们将这些结果解释为氨基酸水合在能量上非常相似，但水分子所处的位置强烈地受到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已发表的氨基酸疏水性的值显著相关。 在离子能量学的第二个领域，我们已经开始研究激光通量和多肽离子碎裂之间的关系。这种类型的研究是优化MALDI TOF/TOF实验以实现肽序列测定的基础。在这些研究中，我们获得了单分子分解和碰撞诱导解离(CID)模式下的多肽碎裂光谱，通常为5000次激光。我们能够很容易地跟踪所考虑的每个肽的两个时间点，即激光激发后1微秒内形成的离子的源内碎裂和仪器碰撞单元内发生的更长的、质量相关的碎裂。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)在所有可用的激光能量范围内的碎裂。我们观察到LeuEnk在比desRb低得多的通量下显示出碎裂的开始。然而，更重要的是，我们已经能够证明，作为增加激光通量的函数的碎片形成是作为一组连续的而不是竞争的反应进行的。这对于多肽测序是非常重要的，因为为了从一系列连续的反应中获得全套碎片离子，有必要形成具有足够的内能来支持整个分解链的目标多肽离子。在多肽混合物的情况下，即对多肽进行测序时的真实情况，只有在MADI实验中才能提供所需的内部能量。同时，我们正在利用气相动力学的莱斯-拉姆斯伯格-卡塞尔-马库斯(RRKM)公式来开发这些分解的动力学模型。除了对碎裂进行建模外，计算还将定义多肽离子温度的下限。有了这些信息，我们将第一次能够估计传输到气相离子的激光能量的比例。此外，我们将有一种方法，而不是纯粹的经验主义，来选择和优化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.