Two‐dimensional crystallization of rabbit C‐reactive protein on lipid monolayers

Sui, Sen‐Fang; Liu, Zheng; Li, Wei; Xiao, Caide; Wang, Shaoxiong; Gao, Qiufeng; Zhou, Qiang

doi:10.1016/0014-5793(96)00528-5

Cited by 15 publications

(19 citation statements)

References 44 publications

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“…2؉ and an Anionic Phospholipid Surface. To further explore syt⅐syt and syt⅐membrane interactions, we used a negative-stain EM approach to image C2A-C2B bound to lipid monolayers (35,36,43,44). Consistent with the data in Fig.…”

Section: Resultssupporting

confidence: 59%

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Bai

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Neuronal exocytosis is mediated by Ca 2؉ -triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca 2؉ -sensing protein proposed to regulate fusion pore dynamics via Ca 2؉ -promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca 2؉ -triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca 2؉ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca 2؉ ⅐C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are Ϸ11 nm long and Ϸ11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca 2؉ first drives C2A-C2B⅐membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca 2؉ -mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.C2 domain ͉ oligomerization ͉ calcium ͉ membrane

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Section: Resultssupporting

confidence: 59%

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Bai

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…The monolayer twodimensional crystallization technique has been widely used since its development by Uzgiris and Kornberg (1983). We used the same technique as in our previous work (Qin et al, 1995;Sui et al, 1996;Wang and Sui, 1999;Wang et al, 2000). Briefly, droplets (ϳ15 l) of 0.1 mg/ml rabbit CRP solution containing 5 mM CaCl 2 , 100 mM NaCl, in 20 mM Tris-HCl buffer with a pH value of 7.4 were placed in a small Teflon well (4 mm in diameter and 0.5 mm in depth).…”

Section: Purification Of Rabbit Crpmentioning

confidence: 99%

“…Rabbit C-reactive protein was purified from the acute-phase serum of rabbits by affinity chromatography on phosphorylethanolamine-Sepharose 4B according to the procedure developed by Sui et al (1996). The purity of the protein was confirmed to be over 99% by SDS-PAGE with silver staining.…”

Section: Purification Of Rabbit Crpmentioning

confidence: 99%

Two-Dimensional Assembly of Pentameric Rabbit C-Reactive Proteins on Lipid Monolayers

Wang

Sui

2001

Journal of Structural Biology

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“…Concerning the fact that the interaction of CRP with membrane is a basic mechanism related to CRP's physiological function, to reveal the structural state of membrane-bound CRP is of extreme importance. In our previous work, different types of two-dimensional (2D) crystals of rabbit CRP (rCRP) on lipid monolayers were obtained in different conditions (22)(23)(24). Twodimensional crystals composed either of pentameric rCRP and monomeric rCRP were obtained.…”

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confidence: 99%