A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Vink, Martin; Derr, KD; Love, J.; Stokes, David L.; Ubarretxena-Belandia, Iban

doi:10.1016/j.jsb.2007.09.003

Cited by 42 publications

(42 citation statements)

References 38 publications

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“…After purification using conditions defined by the New York Consortium on Membrane Protein Structure, we employed a highthroughput approach to systematically test a wide range of parameters relevant to 2D crystallization (16), namely lipid species, lipid:protein ratio, pH, and temperature. A 96-well dialysis block was used to remove detergent from each of the conditions (17), and the samples were negatively stained and screened robotically by EM (18). Narrow tubular crystals formed readily in dioleoylphosphatidyl glycerol (DOPG) after ∼5 d (Fig.…”

Section: Dmentioning

confidence: 99%

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy

Coudray

Valvo

et al. 2013

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

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YiiP is a dimeric Zn 2+ /H + antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn 2+ . Starting from the X-ray structure in the presence of Zn 2+ , we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, fourhelix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.membrane protein | secondary transporter | zinc antiporter | FieF

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Section: Dmentioning

confidence: 99%

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy

Coudray

Valvo

et al. 2013

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

Self Cite

103

134

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“…Three different methods are being used for removing detergents, dialysis, beads, and cyclodextrin 14,31,32 . But it remains difficult to achieve a well-controlled, gradual removal of detergents from a small volume 33,34 . An ideal method for detergent removal would take the detergents out of the aqueous phase evenly across the whole volume in a controllable pace, and should not exert strong interference on the reconstitution of bilayer membranes.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Lee

Zheng

Shi

et al. 2013

JoVE

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To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of welldefined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels. Video LinkThe video component of this article can be found at http://www.jove.com/video/50436/ IntroductionCells exchange materials and information with their environment through the functions of specific membrane proteins 1 . Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate the detrimental effects from the mutant proteins in human diseases.Lipids surround all membrane proteins integrated in bilayers 2,3 . In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5 . Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3,6 . The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins int...

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“…Laboratories are in the process of introducing various degrees of promising automation into the process of screening 8,9 . Yet samples will still require steps of evaluation and knowledge of the 2D crystal size, morphology, and order as outlined here.…”

Section: Discussionmentioning

confidence: 99%

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Johnson

Rudolph

Dreaden

et al. 2010

JoVE

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Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions. By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening. Carbon-coated 400-mesh copper EM grids are prepared by negative stain. Uranyl acetate is frequently used and provides a long-lasting stain in terms of storage of the solution for several months before use as well as suitability for long-term storage of grids. In contrast, other negative stains such as uranyl formate, while providing excellent staining, need to be freshly made 1 . For fast preparation of a large number of grids to be used for screening of 2D crystallization trials, a modified version of negative staining is used. A volume of 2 μL of sample is pipetted onto a carbon-covered EM grid and incubated for 60 s. This is followed by blotting from the edge with a torn piece of Whatman #4 filter paper ( Figure 1; video), and then 2 μL of 1% uranyl acetate are applied immediately, which are again blotted from the edge of the grid after 30 s. Touching the grid on the rim with the torn edge of the filter paper ensures optimal removal of liquid without removal of proteoliposomes. Furthermore, drying at the edge of the grid ensures improved preservation of the carbon film. Care must be taken in preparation and handling of the grids, as breakage of the delicate carbon film prevents sample adhesion and can result in an inaccurate representation of the sample. While traditionally larger sample volumes of 5 μL were and are routinely used for grid preparation, preciou...

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A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Cited by 42 publications

References 38 publications

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

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