Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for me...
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MDPI AG
2022-07-01
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| Series: | Crystals |
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| Online Access: | https://www.mdpi.com/2073-4352/12/7/976 |
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| author | Zhipu Luo Weijie Gu Yichao Wang Yannan Tang Dianfan Li |
| author_facet | Zhipu Luo Weijie Gu Yichao Wang Yannan Tang Dianfan Li |
| author_sort | Zhipu Luo |
| collection | DOAJ |
| description | Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. |
| first_indexed | 2024-03-09T03:32:04Z |
| format | Article |
| id | doaj.art-00e24f345aed4bddbeebbda25827e3e5 |
| institution | Directory Open Access Journal |
| issn | 2073-4352 |
| language | English |
| last_indexed | 2024-03-09T03:32:04Z |
| publishDate | 2022-07-01 |
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| series | Crystals |
| spelling | doaj.art-00e24f345aed4bddbeebbda25827e3e52023-12-03T14:52:40ZengMDPI AGCrystals2073-43522022-07-0112797610.3390/cryst12070976Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic PhaseZhipu Luo0Weijie Gu1Yichao Wang2Yannan Tang3Dianfan Li4Institute of Molecular Enzymology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, ChinaCAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaCAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaCAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaCAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaHeavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP.https://www.mdpi.com/2073-4352/12/7/976crystal soakingexperimental phasinglipid cubic phasemembrane proteinselenourea |
| spellingShingle | Zhipu Luo Weijie Gu Yichao Wang Yannan Tang Dianfan Li Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase Crystals crystal soaking experimental phasing lipid cubic phase membrane protein selenourea |
| title | Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase |
| title_full | Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase |
| title_fullStr | Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase |
| title_full_unstemmed | Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase |
| title_short | Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase |
| title_sort | selenourea for experimental phasing of membrane protein crystals grown in lipid cubic phase |
| topic | crystal soaking experimental phasing lipid cubic phase membrane protein selenourea |
| url | https://www.mdpi.com/2073-4352/12/7/976 |
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