The mechano-sensitive ion channel of M. tuberculosis is a prototypical ion channel of known structure. Despite extensive research efforts, the mechanism by which ion channels conduct ions in response to an activating stimulus is poorly understood. It is the goal of this research to use chemically-synthetic access to the family of mechano-sensitive channels to determine the physical mechanism of channel assembly, conduction and gating. Working in a program project will uniquely complement our chemical synthetic skills with premier ion channel assay expertise. The long-term aim of this research is twofold. Firstly, the development of general, robust methods for the chemical synthesis, biophysical study and manipulation of small membrane proteins. Secondly, to gain unprecedented insight into the mechanistic aspects of ion channel conduction and gating in general. Such understanding will contribute to the design and evaluation of more effective drugs targeted to ion channels. The specific studies envisioned in this proposal can be divided into four categories: 1. Robust total chemical synthesis and reconstitution of MscL (151 amino acids, 16 Kd) 2. Thermodynamics of channel assembly 3. Dynamic structure-function study of channel gating 4. Channels with novel properties In each of these areas we will apply the tools of chemistry to membrane proteins in hitherto unprecedented ways. The understanding arrived at will be tested by the synthesis of non-native ion channel constructs, and channels with modified gating properties as well as novel selectivities.
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