Producing selenomethionine-labeled proteins with a baculovirus expression vector system

Bellizzi, John J.; Widom, Joanne; Kemp, Christopher W.; Clardy, Jon

doi:10.1016/s0969-2126(00)80020-9

Cited by 59 publications

(55 citation statements)

References 24 publications

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“…Very high merging R-factors from multiple crystals indicated that native crystals were non-isomorphous to each other, making the success of isomorphous replacement phasing unlikely. After a systematic study of growth conditions, we were able to express SeMetlabeled PPT1 in the baculovirus system with sufficient incorporation of SeMet to solve the structure by multiwavelength anomalous diffraction phasing (15).…”

Section: Resultsmentioning

confidence: 99%

“…Native and SeMet-labeled bovine PPT1 for the structure determination were overexpressed in insect cells as reported (7,15). PPT1 was secreted into the cell medium.…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme was originally isolated by virtue of its ability to remove palmitate from the proto-oncoprotein H-Ras (7,8), but it has a broad substrate specificity, cleaving fatty acyl chains of lengths [14][15][16][17][18] carbons from cysteine residues in proteins and peptides as well as from palmitoyl-CoA (8) and S-palmitoyl thioglucoside (9). Palmitoylation is an important protein modification that facilitates membrane localization and protein-protein interactions (10).…”

Section: Nfantile Neuronal Ceroid Lipofuscinosis (Incl) Is the Mostmentioning

confidence: 99%

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The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

Bellizzi

Widom

Kemp

et al. 2000

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

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143

125

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Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an ␣͞␤-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.

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Section: Resultsmentioning

confidence: 99%

“…Native and SeMet-labeled bovine PPT1 for the structure determination were overexpressed in insect cells as reported (7,15). PPT1 was secreted into the cell medium.…”

Section: Methodsmentioning

confidence: 99%

Section: Nfantile Neuronal Ceroid Lipofuscinosis (Incl) Is the Mostmentioning

confidence: 99%

See 1 more Smart Citation

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

Bellizzi

Widom

Kemp

et al. 2000

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

Self Cite

143

125

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“…Polyhedra were produced and purified according to established protocols. Crystals from selenomethionine polyhedrin (27) diffracted more consistently and to a higher resolution than the corresponding native polyhedra. Polyhedra were also purified from infected larvae of porina moths (Wiseana spp.)…”

Section: Methodsmentioning

confidence: 99%

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses

Coulibaly

Chiu

Gutmann

et al. 2009

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

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Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the Nterminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be ␣-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-Å structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.in vivo crystallization ͉ molecular arms ͉ occlusion body ͉ self-assembly ͉ virus evolution

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“…In eukaryotes, although considerable progress has been made, incorporation of selenomethionine remains non-trivial (Chen & Bahl, 1991;Lustbader et al, 1995;Bellizzi et al, 1999;McWhirter et al, 1999). Once expressed, selenomethioninelabelled protein is subject to oxidation, which can affect both protein folding and crystallization.…”

Section: Introductionmentioning

confidence: 99%

Selling candles in a post-Edison world: phasing with noble gases bound within engineered sites

Quillin

Matthews

2003

Acta Crystallogr D Biol Cryst

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The utility of noble gases for phase determination has been limited by the lack of naturally occurring binding sites in proteins. Wild-type T4 lysozyme contains one such binding site. By mutating large hydrophobic residues to alanine, additional noble-gas binding sites have been successfully introduced into this protein. Using data from xenon derivatives of the wild type, two single mutants and the corresponding double mutant, experimental phases for T4 lysozyme have been determined using standard multiple isomorphous replacement (MIR) techniques. These phases, which were obtained from room-temperature data collected on a rotating-anode source, are comparable in quality with phases calculated using selenomethionine-based multiwavelength anomalous dispersion (MAD) methods on frozen crystals at a synchrotron. In addition, this method of introducing noble-gas binding sites near speci®c residues should provide useful information for determining the register of amino acids within electron-density maps and the positions of molecules within the unit cell.

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Producing selenomethionine-labeled proteins with a baculovirus expression vector system

Cited by 59 publications

References 24 publications

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses

Selling candles in a post-Edison world: phasing with noble gases bound within engineered sites

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