Design, Synthesis, and Performance of NTA-modified Lipids as Templates for Histidine-tagged Protein Crystallization

Thompson, David H.; Zhou, Mingkang; Grey, Jessica; Jeong, Byung‐Hoon

doi:10.1246/cl.2007.956

Cited by 14 publications

(13 citation statements)

References 139 publications

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“…Interfacial templating strategies using various organic (e.g., lipid monolayers [51][52][53][54], lipid tubules [55] or cubic phases [36]) and inorganic substrates (e.g., gold or mica) have been developed for controlled nucleation of two-dimensional protein crystals (for a review of this and other interfacial approaches see ref. [54]). Mirkin and coworkers have demonstrated the directed growth of polypeptide single crystals using dip pen nanolithography (DPN) [56].…”

Section: Recent Advances and Opportunities In Macromolecular Crystallmentioning

confidence: 99%

Challenges and opportunities for new protein crystallization strategies in structure-based drug design

Grey

Thompson

2010

Expert Opinion on Drug Discovery

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Structure-based drug design (SBDD) has emerged as a valuable pharmaceutical lead discovery tool, showing potential for accelerating the discovery process, while reducing developmental costs and boosting potencies of the drug that is ultimately selected. SBDD is a iterative, rational, lead compound sculpting process that involves both the synthesis of new derivatives and the evaluation of their binding to the target structure either through computational docking or elucidation of the target structure as a complex with the lead compound. This method heavily relies on the production of high-resolution (< 2Å) three-dimensional structures of the drug target, obtained through X-ray crystallographic analysis, in the presence or absence of the drug candidate. The lack of generalized methods for high quality crystal production is still a major bottleneck in the process of macromolecular crystallization. This review provides a brief introduction to SBDD and describes several macromolecular crystallization strategies, with an emphasis on advances and challenges facing researchers in the field today. Recent trends in the development of more universal macromolecular crystallization techniques, particularly nucleation-based techniques that are applicable to both soluble and integral membrane proteins, are also discussed.

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Section: Recent Advances and Opportunities In Macromolecular Crystallmentioning

confidence: 99%

Challenges and opportunities for new protein crystallization strategies in structure-based drug design

Grey

Thompson

2010

Expert Opinion on Drug Discovery

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“…Lipid monolayers have been used to concentrate proteins at interfaces for structure determination, as first described by Uzgiris and Kornberg . Subsequent affinity monolayer developments explored a variety of affinity lipid–ligand interactions such as biotin–streptavidin, ATP lipid–ATP binding protein, Ni 2+ :nitrilotriacetic acid (NTA)-hexahistidine-tagged (His 6 -tag) protein, or Cu 2+ :iminodiacetic acid-His 6 -tag protein . Many of these efforts were focused on the development of two-dimensional crystallization methods using nitrilotriacetic acid (NTA)-modified lipid conjugates, − with some cases showing the potential of NTA lipids for structure elucidation with resolutions in the 10–25 Å regime. ,, NTA lipid monolayers were extended to the realm of cryoEM single particle reconstruction by Walz and co-workers, with the structures for 50S ribosomal subunits, transferrin–transferrin receptor complex, , and human RNA polymerase II reported using a commercially available NTA lipid conjugate, 1,2-dioleoyl- sn -glycero-3-[(( N -(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA), in mixed monolayers with DLPC.…”

Section: Introductionmentioning

confidence: 99%

“…We report our attempts to address these challenges by depositing nonfouling PEG-lipid − coatings containing common affinity ligands like NTA ,,,, onto TEM grids . These coatings are prepared by compression of lipid films whose NTA surface density and PEG mushroom-brush conformational state can be controlled by initial film composition and applied surface pressures prior to Langmuir−Schaefer (LS) deposition onto the EM grid.…”

Section: Introductionmentioning

confidence: 99%

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates

et al. 2016

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We report the preparation and performance of TEM grids bearing stabilized non-fouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol)2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10′,12′-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE) – in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit non-specific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking non-specific adsorption and limiting film degradation, even upon prolonged detergent exposure.

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“…To date only a few efforts of this type have been reported. 17 Such research efforts can be difficult to pursue because they require both expertise in the synthesis and characterization of organic molecules and expertise in the biochemistry of membrane proteins, skill sets that generally do not overlap.…”

Section: Introductionmentioning

confidence: 99%

Crystallographic Characterization of N-Oxide Tripod Amphiphiles

2010

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Tripod amphiphiles are designed to promote the solubilization and stabilization of intrinsic membrane proteins in aqueous solution; facilitation of crystallization is a long-range goal. Membrane proteins are subjects of extensive interest because of their critical biological roles, but proteins of this type can be difficult to study because of their low solubility in water. The nonionic detergents that are typically used to achieve solubility can have the unintended effect of causing protein denaturation. Tripod amphiphiles differ from conventional detergents in that the lipophilic segment contains a branchpoint, and previous work has shown that this unusual amphiphilic architecture can be advantageous relative to traditional detergent structures. Here we report the crystal structures of several tripod amphiphiles that contain an N-oxide hydrophilic group. The data suggest that tripods can adapt themselves to a nonpolar surface by altering the hydrophobic appendage that projects toward that surface and their overall orientation relative to that surface. Although it is not possible to draw firm conclusions regarding amphiphile association in solution from crystallographic data, trends observed among the packing patterns reported here suggest design strategies to be implemented in future studies.

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Design, Synthesis, and Performance of NTA-modified Lipids as Templates for Histidine-tagged Protein Crystallization

Abstract: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Cited by 14 publications

References 139 publications

Challenges and opportunities for new protein crystallization strategies in structure-based drug design

Challenges and opportunities for new protein crystallization strategies in structure-based drug design

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates

Crystallographic Characterization of N-Oxide Tripod Amphiphiles

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