The Extent of Pyrene Excimer Fluorescence Emission Is a Reflector of Distance and Flexibility: Analysis of the Segment Linking the LDL Receptor-Binding and Tetramerization Domains of Apolipoprotein E3

被引:162
作者
Bains, Gursharan K. [1 ]
Kim, Sea H. [1 ]
Sorin, Eric J. [1 ]
Narayanaswami, Vasanthy [1 ]
机构
[1] Calif State Univ Long Beach, Dept Chem & Biochem, Long Beach, CA 90840 USA
基金
美国国家卫生研究院;
关键词
CARBONIC-ANHYDRASE-II; CARBOXYL-TERMINAL DOMAINS; HUMAN PLASMA FIBRONECTIN; ESCHERICHIA-COLI; LACTOSE PERMEASE; APOLIPOPHORIN-III; EXCHANGEABLE APOLIPOPROTEIN; AQUEOUS-SOLUTION; LIPOPROTEIN PREFERENCES; PROXIMITY RELATIONSHIPS;
D O I
10.1021/bi3005285
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375-405 nm) and an additional band (called excimer) at similar to 460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an alpha-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys residues separated by similar to 5 angstrom increments on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were similar to 5 angstrom from each other with an e/m ratio of similar to 3.0, which decreased to similar to 1.0 at 20 angstrom. An inverse correlation between elm ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 A (4.9 angstrom from NMR and 5.7 A from the X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment, suggesting spatial proximity (10-15 angstrom) to corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe for studying protein spatial organization.
引用
收藏
页码:6207 / 6219
页数:13
相关论文
共 68 条
[1]   Structure and mechanism of the lactose permease of Escherichia coli [J].
Abramson, J ;
Smirnova, I ;
Kasho, V ;
Verner, G ;
Kaback, HR ;
Iwata, S .
SCIENCE, 2003, 301 (5633) :610-615
[2]  
AGGERBECK LP, 1988, J BIOL CHEM, V263, P6249
[3]   Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain [J].
Akke, M ;
Liu, J ;
Cavanagh, J ;
Erickson, HP ;
Palmer, AG .
NATURE STRUCTURAL BIOLOGY, 1998, 5 (01) :55-59
[4]   Pyrene: A Probe to Study Protein Conformation and Conformational Changes [J].
Bains, Gursharan ;
Patel, Arti B. ;
Narayanaswami, Vasanthy .
MOLECULES, 2011, 16 (09) :7909-7935
[5]   HELANAL: A program to characterize helix geometry in proteins [J].
Bansal, M ;
Kumar, S ;
Velavan, R .
JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS, 2000, 17 (05) :811-819
[6]   HUMAN PLASMA FIBRONECTIN STRUCTURE PROBED BY STEADY-STATE FLUORESCENCE POLARIZATION - EVIDENCE FOR A RIGID OBLATE STRUCTURE [J].
BENECKY, MJ ;
KOLVENBACH, CG ;
WINE, RW ;
DIORIO, JP ;
MOSESSON, MW .
BIOCHEMISTRY, 1990, 29 (12) :3082-3091
[7]   IONIC-STRENGTH-DEPENDENT AND PH-DEPENDENT CONFORMATIONAL STATES OF HUMAN PLASMA FIBRONECTIN [J].
BENECKY, MJ ;
WINE, RW ;
KOLVENBACH, CG ;
MOSESSON, MW .
BIOCHEMISTRY, 1991, 30 (17) :4298-4306
[8]   Triplet-triplet interaction of aromatic molecules in solution [J].
Blrks, J. B. .
CHEMICAL PHYSICS LETTERS, 1968, 2 (06) :417-419
[9]  
Bunau G. V., 1970, JB BIRKSPHOTOPHYSICS, V74, P1294
[10]   EXCIMER FLUORESCENCE OF EQUINE PLATELET TROPOMYOSIN LABELED WITH N-(1-PYRENYL)IODOACETAMIDE [J].
BURTNICK, LD ;
STEWART, DIH ;
CLARK, ID ;
SMILLIE, LB .
BIOCHEMISTRY, 1986, 25 (13) :3875-3880