Type of Document	Dissertation
Author	Mo, Yiming
Author's Email Address	laomo3@yahoo.com
URN	etd-12212005-141748
Title	Study of Structure-Function Correlations in Ion Channels by Solid State NMR
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Timothy A. Cross Committee Chair Piotr G. Fajer Committee Member Randolph Rill Committee Member Timothy M. Logan Committee Member William Cooper Committee Member
Keywords	Ion Channel Membrane Protein NMR
Date of Defense	2005-12-14
Availability	unrestricted
Abstract Structural biology of membrane proteins and ion channels are of great interest because of their important roles in many cellular and physiological processes including ion/molecular transport, communication and energy transduction. However, only a limited number of high resolution structures have been published because membrane proteins and ion channels are rich in highly hydrophobic segments with poor aqueous solubility, and require lipid environments to maintain their functional structure. Apart from x-ray crystallography, EPR, solution NMR, and electron diffraction, solid state NMR is an alternative method for the study of structural/functional correlations. Structures of many transmembrane domains (TMDs, peptides) of membrane proteins have been solved by solid state NMR with TMDs incorporated in uniformly aligned lipid bilayers. To apply that method to full length membrane protein, additional factors should be considered for sample preparation, such as isotopical labeling, sample stability and experimental sensitivity. To optimize the conditions for oriented sample preparation, M2 protein from Influenza A is used. 15N-Leu specific labeled M2 protein have been overexpressed in E. Coli, purified and reconstituted into lipid bilayers. Lipid bilayers were uniformly aligned on glass slides. The orientation of M2 protein was monitored by 15N-NMR. Several parameters were used in sample preparation: composition of lipid bilayers, hydration level, protein-lipid molar ratio, and detergents. Changing composition of lipid bilayers from DMPC/DMPG/DMPE, POPC/POPG/POPE, to DOPC/DOPG/DOPE with different hydrophobic thickness has little effect on the alignment of M2 protein. High water content in the samples induced serious RF heating in the samples, resulting in sample inhomogeneity. Protein-lipid molar ratios from 1:50 to 1:200 have no effect on in protein alignment. The most important parameter is the detergent used in sample preparation. Samples prepared by using a strong detergent such as SDS resulted in 80% of the M2 protein being aligned while use of a weak detergent like OG resulted in only 40%. The NMR spectra also support a tilt angle of 20 „b 5„a for the transmembrane domain. The ion interactions with KcsA, a potassium channel from Streptomyces lividans were studied by 87Rb NMR. KcsA was overexpressed in E. Coli, purified and reconstituted in DOPC/DOPG (4:1) liposomes. The 87Rb NMR study supported a two-step model for how ions enter the channel from bulk solution. The rubidium ions bind to the lipid bilayers first and then the lipid bound rubidium ions transfer to the proteinˇ¦s pore region of the channel. The rate of first step is slow but greater than 10 Hz. The second step is faster (>> 300 Hz). The binding constant of rubidium ions to KcsA was estimated to be 65 M-1 when [Rb+] = 2 mM, by assuming a first order binding process. Such a mechanism suggests a novel way for Ca2+ to inhibit the channel function. Potentially, by depleting the surrounding lipid of Rb+ or K+, Ca2+ may inhibit conductance.
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