Electron Microscopic Evidence in Support of α-Solenoid Models of Proteasomal Subunits Rpn1 and Rpn2

被引:34
作者
Effantin, Gregory [1 ]
Rosenzweig, Rina [2 ]
Glickman, Michael H. [2 ]
Steven, Alasdair C. [1 ]
机构
[1] NIAMSD, Struct Biol Res Lab, NIH, Bethesda, MD 20892 USA
[2] Technion Israel Inst Technol, Dept Biol, IL-32000 Haifa, Israel
基金
以色列科学基金会;
关键词
negative staining electron microscopy; image classification; circular dichroism; alpha-solenoids; PROTEIN SECONDARY STRUCTURE; CIRCULAR-DICHROISM SPECTRA; REGULATORY PARTICLE; 26S PROTEASOME; CRYOELECTRON MICROSCOPY; S-PROTEASOME; COMPLEX; BINDS; YEAST; VISUALIZATION;
D O I
10.1016/j.jmb.2009.01.039
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
Rpn1 (109 kDa) and Rpn2 (1.04 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal alpha-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each similar to 40 residues long and containing two alpha-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly alpha-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the alpha-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures. Published by Elsevier Ltd.
引用
收藏
页码:1204 / 1211
页数:8
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