Author:
Lee Ho-Hsien,Cherni Irene,Yu HongQi,Fromme Raimund,Doran Jeffrey D.,Grotjohann Ingo,Mittman Michele,Basu Shibom,Deb Arpan,Dörner Katerina,Aquila Andrew,Barty Anton,Boutet Sébastien,Chapman Henry N.,Doak R. Bruce,Hunter Mark S.,James Daniel,Kirian Richard A.,Kupitz Christopher,Lawrence Robert M.,Liu Haiguang,Nass Karol,Schlichting Ilme,Schmidt Kevin E.,Seibert M. Marvin,Shoeman Robert L.,Spence John C. H.,Stellato Francesco,Weierstall Uwe,Williams Garth J.,Yoon Chunhong,Wang Dingjie,Zatsepin Nadia A.,Hogue Brenda G.,Matoba Nobuyuki,Fromme Petra,Mor Tsafrir S.
Abstract
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein ofHuman immunodeficiency virus 1(HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed inEscherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
Funder
National Institutes of Health
National Science Foundation
U.S. Department of Energy
Max-Planck-Gesellschaft
Helmholtz Association
Publisher
International Union of Crystallography (IUCr)
Subject
Condensed Matter Physics,General Materials Science,Biochemistry,General Chemistry
Cited by
6 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献