The Affinity Grid: A Pre-fabricated EM Grid for Monolayer Purification

Kelly, Deborah F.; Abeyrathne, Priyanka D.; Dukovski, Danijela; Walz, Thomas

doi:10.1016/j.jmb.2008.07.023

Cited by 80 publications

(88 citation statements)

References 13 publications

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“…Affinity Capture devices can withstand some degree of detergents for a brief period of time, although this needs to be determined on a case-by-case basis. Please refer to the list of compatible reagents previously described 22 .…”

Section: Discussionmentioning

confidence: 99%

<em>In situ</em> TEM of Biological Assemblies in Liquid

Dukes

Gilmore

Tanner

et al. 2013

JoVE

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Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments-viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

<em>In situ</em> TEM of Biological Assemblies in Liquid

Dukes

Gilmore

Tanner

et al. 2013

JoVE

Self Cite

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“…GST-SYT-C2AB was expressed in Escherichia coli BL21(DE3) and was purified as previously described (10). EM on lipid monolayers was carried out as described (29). Briefly, lipid monolayers formed at the air/water interface were recovered by a carbon-coated EM grid (400 mesh; Ted Pella).…”

Section: Methodsmentioning

confidence: 99%

“…To obtain oligomeric SYT-C2AB domains on lipid surfaces, we adapted the conditions used for 2D crystallization of protein kinase C (28), combined with the affinity grid technique (29). Briefly, the lipid monolayer formed at the air/water interface was recovered by placing a carbon-coated EM grid on top of the lipid hydrophobic tails.…”

Section: Syt-c2ab Domains Oligomerize As Circular Structures On the Mmentioning

confidence: 99%

Calcium sensitive ring-like oligomers formed by synaptotagmin

Bello¹,

Auclair²,

Wang³

et al. 2014

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

159

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The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear.Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. S ynaptotagmin-1 (SYT) is the calcium (Ca 2+ ) sensor that triggers synchronous release of neurotransmitters for synaptic transmission (1-4). It is a transmembrane protein, localized to the synaptic vesicles (1, 5), with tandem cytosolic C2 domains (C2A and C2B) that bind phospholipids in both a Ca 2+ -independent and a Ca 2+ -dependent manner (1, 6). The membrane-distal C2B domain interacts with acidic lipids such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to mediate efficient docking of the synaptic vesicles (7-10) before the influx of Ca 2+ ions. Recent studies (7,8,11,12) have located this calciumindependent membrane-binding site to a polybasic patch on the C2B domain (site I in Fig. 1A). The binding of Ca 2+ to the calcium-coordination pocket of the C2B domain (site II in Fig. 1A) triggers the insertion of flanking portions of site II into the plasma membrane, and this Ca 2+ -triggered membrane insertion is absolutely required for neurotransmitter release (13-16).The C2B domain also mediates the Ca 2+ -independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17-21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22). How the insertion of site II into the membrane bilayer is coupled to the completion of the SNARE assembly to release neurotransmitter is still unclear, although some key points have been established. In the prefusion state, the SNARE complexes...

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“…In this type of process, a monolayer of nickel-functionalized polybutadiene-polyethylene oxide (PB-PEO) is collected at the water-interface, and includes the presence of aqueous solution, histidinetagged AqpZ, PDMS-PMOXA-PDMS, and mixed micelles of detergent [17]. The property of nickel ainity to histidine further connects the AqpZ to the PB-PEO layer [18], efectually creating a dynamic of AqpZ high packing within the layer. Once the detergent is removed with the aid of biobeads and the PB-PEO is taken out with imidazole, the closely packed AqpZ PMOXA-PDMS-PMOXA crystals remained; however, the left over amount was not suicient for retrieving key structural data [19,20].…”

Section: Assessing Aquaporin Proteins and Block Copolymer Matrixes Inmentioning

confidence: 99%

Interactions between Aquaporin Proteins and Block Copolymer Matrixes

Abdelrasoul¹,

Doan²,

Lohi³

2017

Biomimetic and Bioinspired Membranes for New Frontiers in Sustainable Water Treatment Technology

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This chapter continues to further expand its focus on aquaporins (AQPs) by ofering a general outline on how the AQPs block copolymers, and polymer support structures can interrelate and such connections can be comprehensively classiied and deined. The irst section of the overview will consider the relationship between block copolymers and AQPs. It will also examine the general membrane protein integration into block copolymers, since this can cause AQP-block copolymer complexes in vesicular (proteopolymersomes) as well as in planar forms. The majority of considerations taken into account during AQP incorporation come from the research conducted in relation to the process of incorporating other types of membrane proteins. This chapter includes an overview of the various characterization methodologies needed for the study of proteopolymersomes, as well as freeze-fracture transmission electron microscopy (FF-TEM), luorescence correlation spectroscopy (FCS), small-angle X-ray scatering (SAXS), and stopped-low light scatering (SFLS). The research data presented in this chapter emphasizes the fact that a successful process of membrane fabrication requires the integration of reconstituted AQPs into a suitable supporting matrix formation.Keywords: aquaporin proteins, block copolymer, matrix, vesicular, membrane protein Assessing aquaporin proteins and block copolymer matrixes interactionsMost research performed on membrane protein inclusion has been conducted primarily with lipids as the host matrix components (original proteoliposomes publication on the subject came out in 1971) [1]. Since then, polymer-based incorporation process has received increased atention and the earliest proteopolymersomes publication emerged in 2000 [2]. The initial work stages in this research area concentrated on the inclusion of membranespanning proteins, such as membrane-bound ion channels (ATPases) and bacteriorhodopsin incorporation into polymethyloxazoline-polydimethylsiloxane-polymethyloxazoline (PMOXA-PDMS-PMOXA) triblock copolymer bilayers occurring in planar [3] or vesicular forms [4][5][6]. It is quite fascinating that the membrane proteins may be functionally incorporated into polymeric bilayers (e.g., based on PMOXA-PDMS-PMOXA) that occur up to 10 times thicker than their lipidic counterparts [7]. Moreover, researchers have observed proteopolymersomes with protein density values that drastically surpassed those of proteoliposomes [8]. Research on these phenomena has helped to establish a theoretical approach for generalized membrane protein incorporation into the amphiphilic structures. This methodology is based on the notion that the eiciency of the membrane protein incorporation process relies on its hydrophobicity potential and its coupling capacity to the host membrane is directly connected to the hydrophobic mismatch parameters. In order to reduce this mismatch dynamic, the method calls for the host membrane to be deformed in such a way that it matches the hydrophobic length parameter of the membrane protein's transm...

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The Affinity Grid: A Pre-fabricated EM Grid for Monolayer Purification

Cited by 80 publications

References 13 publications

<em>In situ</em> TEM of Biological Assemblies in Liquid

<em>In situ</em> TEM of Biological Assemblies in Liquid

Calcium sensitive ring-like oligomers formed by synaptotagmin

Interactions between Aquaporin Proteins and Block Copolymer Matrixes

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