Controlled 2D crystallization of membrane proteins using methyl-β-cyclodextrin

Signorell, Gian A.; Kaufmann, Thomas C.; Kukulski, Wanda; Engel, Andréas; Rémigy, Hervé W.

doi:10.1016/j.jsb.2006.07.011

Cited by 82 publications

(68 citation statements)

References 30 publications

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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 .…”

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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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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“…Classical approaches to reconstitute membrane proteins into liposomes include different methods of controlled detergent removal, such as dilution, dialysis, Biobeads, gel filtration and cyclodextrins. [15][16][17] Removal of the stabilizing detergents promotes the interaction of membrane proteins with the respective lipids or block copolymers, eventually yielding protein reconstituted into liposomes or polymerosomes. Reconstitution can occur either from protein-lipid-detergent ternary complexes with detergent solubilized lipids or from solubilized protein and preformed liposomes that are destabilized upon addition of detergent.…”

Section: Vision and Conclusionmentioning

confidence: 99%

Engineering and Assembly of Protein Modules into Functional Molecular Systems

Hirschi

Stauffer

Harder

et al. 2016

Chimia

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Synthetic biology approaches range from the introduction of unique features into organisms to the assembly of isolated biomacromolecules or synthetic building blocks into artificial biological systems with biomimetic or completely novel functionalities. Simple molecular systems can be based on containers on the nanoscale that are equipped with tailored functional modules for various applications in healthcare, industry or biological and medical research. The concept, or vision, of assembling native or engineered proteins and/ or synthetic components as functional modules into molecular systems is discussed. The main focus is laid on the engineering of energizing modules generating chemical energy, transport modules using this energy to translocate molecules between compartments of a molecular system, and catalytic modules (bio-)chemically processing the molecules. Further key aspects of this discourse are possible approaches for the assembly of simple nanofactories and their applications in biotechnology and medical health.

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“…Over the past years the methodology has seen major advances. While the best 2D crystals are still being produced by dialysis [17], new ways of producing 2D crystals of membrane proteins have been introduced [18][19][20]. Still lacking are 2D crystallization screens as those available for 3D crystallization, but efforts to establish such screens are currently underway.…”

Section: The Time It Takes To Solve a Structurementioning

confidence: 99%

Revival of electron crystallography

Hite

Raunser

Walz

2007

Current Opinion in Structural Biology

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SummarySince the structure determination of bacteriorhodopsin in 1990, much progress has been made in the further development and use of electron crystallography. In this review, we provide a concise overview of the new developments in electron crystallography concerning 2D crystallization, data collection and data processing. Based on electron crystallographic work on bacteriorhodopsin, the acetylcholine receptor and aquaporins, we highlight the unique advantages and future perspectives of electron crystallography for the structural study of membrane proteins. These advantages include the visualization of membrane proteins in their native environment without detergent-induced artifacts, the trapping of different states in a reaction pathway by time-resolved experiments, the study of non-specific protein-lipid interactions and the characterization of the charge state of individual residues in membrane proteins. The structuresElectron crystallography began with Henderson and Unwin's groundbreaking work on the structure of purple membrane. Introducing glucose embedding as a new specimen preparation method and electron crystallographic data processing, these pioneers could produce a density map of bacteriorhodopsin (bR) at 7Å resolution [1,2]. The map resolved for the first time membrane-spanning α-helices and provided the first view of the structural organization of a membrane protein. After improvements in specimen preparation, data collection and data processing, Henderson and co-workers could finally present an atomic model of bR, the first structure solved by electron crystallography [3]. Since then, a higher resolution map of bR was obtained [4] and the structures of six more membrane proteins were solved by electron crystallography: plant light-harvesting complex 2 [5], aquaporin-1 [6,7], torpedo ray nicotinic acetylcholine receptor [8,9], sheep aquaporin-0 [10,11••], rat aquaporin-4 (solved using recombinant protein expressed in insect cells) [12••] and rat microsomal glutathione transferase 1 [13•]. A game of numbers The number of solved structuresSeven structures may appear a modest accomplishment compared to the many more membrane protein structures that have been determined by x-ray crystallography over the past few years. Considering, however, that less than two dozen groups are currently pursuing electron crystallography, compared to the hundreds of x-ray groups, the seven membrane protein structures determined by electron crystallography compare quite favorably to those determined by x-ray crystallography. More importantly, however, unlike x-ray crystallography, electronCorresponding author: Walz, Thomas (twalz@hms.harvard.edu) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process...

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Controlled 2D crystallization of membrane proteins using methyl-β-cyclodextrin

Cited by 82 publications

References 30 publications

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

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

Engineering and Assembly of Protein Modules into Functional Molecular Systems

Revival of electron crystallography

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