A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

1988 ◽  
Vol 46 ◽  
pp. 152-153 ◽  
Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi
Keyword(s):  
Membrane Proteins ◽  
Flow Rate ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Dimensional Structure ◽  
Electron Crystallography ◽  
Peltier Element ◽  
Protein Ratio ◽  
Precise Control ◽  
3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

scholarly journals Membrane proteins: always an insoluble problem?

2016 ◽  
Vol 44 (3) ◽  
pp. 790-795 ◽  
Author(s):  
Andrea E. Rawlings
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Drug Targets ◽  
Functional Characterization ◽  
Water Soluble ◽  
High Yield ◽  
Cellular Processes ◽  
Native Sequence ◽  
Protein Research ◽  
New Methodologies

Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence.

Structure and dynamics of polypeptides and proteins in lipid membranes

1992 ◽  
Vol 25 (4) ◽  
pp. 433-457 ◽  
Author(s):  
Horst Vogel
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Molecular Mechanisms ◽  
Purple Bacteria ◽  
Structural Data ◽  
Transport Processes ◽  
Dimensional Structure ◽  
Expression Cloning ◽  
Biological Research ◽  
Hydrophobic Barrier

The elucidation of the molecular mechanisms whereby ions and polar molecules are translocated across the hydrophobic barrier of a lipid bilayer in biological membranes is one of the most challenging problems in biological research. Specific membrane proteins, such as pumps, carriers and channels, play the central role in the various translocation pathways. Recent progress in expression cloning has provided the sequence of a number of biologically important membrane proteins and in principle the door is open to investigate every protein which might be of importance in the central signal transduction and transport processes. Unfortunately, to date there are only a few examples where the three-dimensional structure of membrane proteins are known at atomic resolution. The photosynthetic reaction centres from purple bacteria (Deisenhoferet al.1985), bacteriorhodopsin (Hendersonet al.1990) and the large porin channel ofRhodobacter capsulata(Weisset al.1991). According to these structural data membrane proteins seem to fold in general in membrane-spanning α-helices and β-strands in order to saturate hydrogen bonds. Only these two motifs seem to form stable structures which can be in contact with the hydrophobic lipid interior of a membrane.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

scholarly journals 3-Dimensional Structure-Imitation Model of Evolution of Microstructure of Powder Body During Sintering

1999 ◽  
Vol 32 (1-4) ◽  
pp. 221-233
Author(s):  
I. G. Kamenin ◽  
R. M. Kadushnikov ◽  
V. M. Alievsky ◽  
D. M. Alievsky ◽  
S. V. Somina
Keyword(s):  
Powder Compact ◽  
3D Structure ◽  
Particle Interaction ◽  
Spherical Particles ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Powder Body ◽  
Time Dependencies ◽  
Growth Laws ◽  
Evolution Of Microstructure

This paper describes a 3D structure-imitation computer model of evolution of the powder compact during sinteringand recrystallization without nucleation. At the initial stages of the evolution processes (sintering until a mosaic structure of boundaries is formed) the model particles are spheres, and two-particle interaction laws control their evolution. During sintering the degree of mutual penetration of the particles increases, regions where spherical particles are wholly facetted by contacts with neighboring particles are formed and grow. These particles are described using the formalism of Voronoi radical polyhedra, and grain growth laws govern their evolution. The model predicts the time dependencies of the following structure parameters of the polyhedra: average polyhedron size and dispersion, total surface of the facets of the polyhedra and total lenght of the edges of the polyhedra.

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