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Theoretical Studies On The Dynamic Aspects Of Macromolecular Function

大分子功能动态方面的理论研究

基本信息

批准号：
7734018
负责人：
Attila Szabo
金额：
$ 30.36万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Membrane channels that allow metabolites to exchange between cells or different cellular compartments are protein structures with water-filled pores. Traditionally such channels were thought to be molecular sieves which discriminate between different solutes based only on size. In other words, they were regarded as low-selectivity structures that allow passage of the solute without any significant interaction with the channel pore. We have shown how nature can in principle exploit and tune the solute-pore interaction in such a way to maximize the solute flux(i.e., the number of molecules translocated per second) and therefore increase the efficiency of the channel. The basic idea is that when only one solute can be in the channel, the flux must have a maximum as a function of the solute-channel interaction. Physically the reason is the following.If the interaction is too attractive, the solute will never leave the channel thus blocking it for the passage of other molecules. If it is too repulsive, the solute molecule will never enter the channel. Since the flux vanishes in both these limits, there must exist an optimum interaction for which the transport is the fastest. We have constructed a quantitative mathematical model for this process and using the calculus of variations determined the optimal intrachannel interaction potential that maximizes the flux. This optimum potential turns out to be tilted towards the side with lower concentration. This makes the counterintuitive prediction that it is better for the solute to "bind" more strongly near the "exit" of the channels rather than near the "entrance". In addtion we found that the value of the optimum interaction potential depends on the concentration od solute outside the channel. This suggests that in a given organism, channel proteins designed to transport the same molecule may have different amino acid sequences. One gene might code for a channel protein that functions at high solute concentrations, while another for one that works at low concentrations. Dramatic advances have been made recently in our ability to study the behavior of single macromolecules. The controlled application of mechanical forces on single molecules has provided a powerful tool to study their structure, function and dynamics. These new experiments require sopisticated cutting-edge technologies and the development of new theoretical approaches to interpret them. Dynamic force spectroscopy probes both kinetics and thermodynamics and a long term interest of this laboratory has been the development of the theory required to analyze such experiments. In order to make the advances we made over the years accessible to a wide audience of potential users, we have written a chapter entitled "Thermodynamics and Kinetics from Single-Molecule Force Spectroscopy" in a book published this year that contains contributions from all the leading research groups throuhout the world. The major contribution we made this year was the development of a novel and simple procedure to extract kinetic information ( specifically the intrinsic rates and free energies of activation) from single molecule pulling experiments performed using optical tweezers and atomic force microscopes. The cornerstone of our method is a transformation ot the rupture-force histograms obtained at different force-loading rates into the force dependent lifetimes measurable in contant-force experiments. To interpret these force dependent lifetimes we derived a generalization ot the widely-used Bell's formula that is exact within the framework of Kramers theory of diffusive barrier crossing. We have illustrated our procedure by analyzing the nanopore unzipping of DNA hairpins and the unfolding of a protein attached by flexible linkers to an atomic force miscroscope. We have shown that our approach remains valid even when the molecular extension is a poor reaction coordinate and higher dimensional free energy surfaces must be considered. We believe that our procedure will become the de facto standard way of interpreting experimental data in this rapidly emerging area.

允许代谢物在细胞或不同细胞间交换的膜通道是具有充满水的孔的蛋白质结构。传统上，这种通道被认为是分子筛，仅根据大小来区分不同的溶质。换句话说，它们被认为是低选择性结构，允许溶质通过而不与通道孔有任何显著的相互作用。我们已经展示了自然界如何在原则上利用和调整溶质-孔隙相互作用，以使溶质通量（即溶质通量）最大化。（即每秒转移的分子数），从而提高通道的效率。基本思想是，当通道中只有一种溶质时，通量作为溶质通道相互作用的函数必须有一个最大值。物理上的原因如下。如果相互作用太有吸引力，溶质就永远不会离开通道，从而阻碍了其他分子的通过。如果斥力太强，溶质分子将永远无法进入通道。由于通量在这两个极限下都消失了，因此必然存在一个输运最快的最佳相互作用。我们为这一过程建立了一个定量的数学模型，并利用变分法确定了使通量最大化的最优通道内相互作用势。结果表明，最佳电位向浓度较低的一侧倾斜。这就产生了一个违反直觉的预测，即溶质在通道“出口”附近比在通道“入口”附近“结合”更强。此外，我们发现最佳相互作用电位的值取决于通道外溶质的浓度。这表明，在给定的生物体中，用于运输相同分子的通道蛋白可能具有不同的氨基酸序列。一个基因可能编码在高溶质浓度下起作用的通道蛋白，而另一个基因编码在低溶质浓度下起作用的通道蛋白。