A novel tool for probing membrane protein structure: Solid-state NMR with proton spin diffusion and X-nucleus detection

被引:98
作者
Kumashiro, KK
Schmidt-Rohr, K [1 ]
Murphy, OJ
Ouellette, KL
Cramer, WA
Thompson, LK
机构
[1] Univ Massachusetts, Dept Polymer Sci & Engn, Amherst, MA 01003 USA
[2] Univ Massachusetts, Dept Chem, Amherst, MA 01003 USA
[3] Univ Massachusetts, Mol & Cellular Biol Program, Amherst, MA 01003 USA
[4] Purdue Univ, Dept Biol Sci, W Lafayette, IN 47907 USA
关键词
D O I
10.1021/ja972655e
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
We present a new solid-state NMR approach, based on H-1 spin diffusion with X-nucleus (N-15, C-13, P-31) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman-Shen H-1 spin-diffusion experiment with X-nucleus detection to proteins in hydrated Lipid bilayers (>25% water by weight)in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H2O molecules, which can be selected efficiently by the H-1 T-2 filter in the Goldman-Shen sequence. The transfer of H-1 magnetization from mobile H2O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide N-15 signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spin-diffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the P-31 in the Lipid phosphate head groups and the C-13 nuclei in the Lipid acyl chains as distance markers for the spin diffusion.
引用
收藏
页码:5043 / 5051
页数:9
相关论文
共 39 条
[31]   DETERMINATION OF HELIX-HELIX INTERACTIONS IN MEMBRANES BY ROTATIONAL RESONANCE NMR [J].
SMITH, SO ;
BORMANN, BJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (02) :488-491
[32]   H-1 SPIN-DIFFUSION COEFFICIENTS OF HIGHLY MOBILE POLYMERS [J].
SPIEGEL, S ;
SCHMIDT-ROHR, K ;
BOEFFEL, C ;
SPIESS, HW .
POLYMER, 1993, 34 (21) :4566-4569
[33]   THE COLICIN E1 INSERTION-COMPETENT STATE - DETECTION OF STRUCTURAL-CHANGES USING FLUORESCENCE RESONANCE ENERGY-TRANSFER [J].
STEER, BA ;
MERRILL, AR .
BIOCHEMISTRY, 1994, 33 (05) :1108-1115
[34]  
VENYAMINOV SY, 1998, IN PRESS P NATL ACAD, V95
[35]  
Wang JX, 1997, BIOCHEMISTRY-US, V36, P1699, DOI 10.1021/bi962578k
[36]  
WANG Y, 1988, INTERFACES POLYM CER, P249
[37]   STRUCTURE OF A FLUID DIOLEOYLPHOSPHATIDYLCHOLINE BILAYER DETERMINED BY JOINT REFINEMENT OF X-RAY AND NEUTRON-DIFFRACTION DATA .3. COMPLETE STRUCTURE [J].
WIENER, MC ;
WHITE, SH .
BIOPHYSICAL JOURNAL, 1992, 61 (02) :434-447
[38]   Membrane binding of the colicin E1 channel: Activity requires an electrostatic interaction of intermediate magnitude [J].
Zakharov, SD ;
Heymann, JB ;
Zhang, YL ;
Cramer, WA .
BIOPHYSICAL JOURNAL, 1996, 70 (06) :2774-2783
[39]   CONSTRAINTS IMPOSED BY PROTEASE ACCESSIBILITY ON THE TRANS-MEMBRANE AND SURFACE-TOPOGRAPHY OF THE COLICIN-E1 ION CHANNEL [J].
ZHANG, YL ;
CRAMER, WA .
PROTEIN SCIENCE, 1992, 1 (12) :1666-1676