scholarly journals Quantifying membrane protein oligomerization using two-dimensional fluorescence intensity fluctuation spectrometry

2019 ◽  
Keyword(s):  
Membrane Protein ◽  
Fluorescence Intensity ◽  
Intensity Fluctuation ◽  
Protein Oligomerization ◽  
Two Dimensional

scholarly journals Quantifying membrane protein oligomerization using two-dimensional fluorescence intensity fluctuation spectrometry

2019 ◽  
Author(s):  
Michael R. Stoneman ◽  
Gabriel Biener ◽  
Richard J. Ward ◽  
John D. Pediani ◽  
Dammar Badu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescence Intensity ◽  
Laser Scanning ◽  
Intensity Fluctuation ◽  
Membrane Receptors ◽  
Protein Oligomerization ◽  
Two Dimensional ◽  
Receptor Concentration ◽  
Standard Laser ◽  
Detailed Protocol

Abstract Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.

scholarly journals Reply To: Molecular Brightness analysis of GPCR oligomerization in the presence of spatial heterogeneity

2019 ◽  
Author(s):  
Michael R. Stoneman ◽  
Gabriel Biener ◽  
Valerică Raicu
Keyword(s):  
Spatial Heterogeneity ◽  
Fluorescence Intensity ◽  
Intensity Fluctuation ◽  
Regions Of Interest ◽  
Cellular Structures ◽  
Two Dimensional ◽  
Analysis Process ◽  
Brightness Analysis ◽  
Molecular Brightness ◽  
Selection Of

Annibale and Lohse have recently suggested a way1 in which the two-dimensional fluorescence intensity fluctuation (2D FIF) spectrometry2 may be further refined. Their main suggestion is to include a step in the analysis process where a case-by-case inspection of individual regions of interest of a membrane allows for selection of portions of the membrane which are “as homogenous as possible” and thereby exclude intensity spots potentially related to other sub-cellular structures. By incorporating that proposal into an objective and reproducible algorithm, here we show that 2D FIF has a built-in capability to automatically filter out such contributions, and that further removal of inhomogeneities does not alter the final results.

scholarly journals Determination of Membrane Protein Transporter Oligomerization in Native Tissue Using Spatial Fluorescence Intensity Fluctuation Analysis

PLoS ONE ◽  
2012 ◽  
Vol 7 (4) ◽  
pp. e36215 ◽  
Author(s):  
Mikhail Sergeev ◽  
Antoine G. Godin ◽  
Liyo Kao ◽  
Natalia Abuladze ◽  
Paul W. Wiseman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescence Intensity ◽  
Intensity Fluctuation ◽  
Fluctuation Analysis ◽  
Native Tissue

Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase

1998 ◽  
Vol 123 (2) ◽  
pp. 87-96 ◽  
Author(s):  
Ingeborg Schmidt-Krey ◽  
Gerd Lundqvist ◽  
Ralf Morgenstern ◽  
Hans Hebert
Keyword(s):  
Membrane Protein ◽  
Glutathione Transferase ◽  
Two Dimensional ◽  
Microsomal Glutathione

scholarly journals Two-Dimensional Fluorescence Intensity Distribution Analysis: Theory and Applications

2000 ◽  
Vol 78 (4) ◽  
pp. 1703-1713 ◽  
Author(s):  
Peet Kask ◽  
Kaupo Palo ◽  
Nicolas Fay ◽  
Leif Brand ◽  
Ülo Mets ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Intensity Distribution ◽  
Distribution Analysis ◽  
Two Dimensional ◽  
Analysis Theory ◽  
Fluorescence Intensity Distribution Analysis

scholarly journals Adición de proteínas del plasma seminal ovino durante la congelación del espermatozoide y efectos sobre su motilidad y viabilidad

2009 ◽  
Vol 10 (1) ◽  
pp. 51 ◽  
Author(s):  
Jaime Antonio Cardozo ◽  
Patricia Grasa ◽  
María Teresa Muiño B. ◽  
José Álvaro Cebrián P.
Keyword(s):  
Membrane Protein ◽  
Sperm Motility ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Plasma Proteins ◽  
Two Dimensional ◽  
Plasma Séminal ◽  
Sperm Membrane ◽  
Seminal Plasma Proteins ◽  
Ram Sperm

<p>Este estudio se adelantó para evaluar el efecto de la adición de proteínas del plasma seminal de cordero en la criopreservación sobre la motilidad e integridad de la membrana espermática, y los cambios en el perfil electroforético de las proteínas de la membrana espermática inducidos por la criopreservación. Se usaron eyaculados de ocho corderos adultos de la raza rasa aragonesa, se les determinó su viabilidad y motilidad espermáticas y posteriormente se sometieron a un procedimiento de congelación. Las proteínas se separaron por el método de electroforesis en geles de acrilamida en dos dimensiones. Se obtuvo un mejoramiento significativo (<em>p </em>&lt; 0,05) en la calidad del semen congelado, cuando se adicionaron proteínas del plasma seminal. El análisis bidimensional comparativo entre el semen fresco y el congelado evidenció la pérdida de 8 puntos de proteína en el espermatozoide descongelado. La concentración de un punto de proteína de membrana espermática, de bajo peso molecular (punto 2), fue más alta (<em>p </em>&lt; 0,05) en el espermatozoide descongelado al que se adicionaron proteínas del plasma seminal. Se encontraron correlaciones entre algunos puntos de proteína y la motilidad y viabilidad espermáticas, lo cual sugiere que pueden jugar papeles importantes en el mantenimiento de la integridad y funcionalidad del espermatozoide. Se puede concluir que la adición de proteínas del plasma seminal en la congelación mejora la integridad del espermatozoide descongelado, y que la criopreservación del semen de cordero produce variaciones en la composición de las proteínas de membrana.  </p><p> </p><p><strong>Effect of seminal plasma proteins at freezing on ram sperm motility and viability</strong>  </p><p>The aim of the study was to evaluate the cryoprotective effect of seminal plasma proteins on ram sperm motility, membrane integrity and the changes in the profile of ram sperm membrane proteins induced by cryopreservation. Fresh ejaculates from 8 mature Rasa aragonesa rams were used. Sperm motility and cell viability was assessed. The freezing procedure was based on the method described by Fiser <em>et al</em>. (1987). Proteins extracted from fresh and frozen-thawed semen were subjected to the Two-dimensional polyacrilamide gel electrophoresis. A significant improvement in the quality of frozenthawed sperm was obtained after addition of seminal plasma proteins (<em>p </em>&lt; 0.05). Comparative two-dimensional polyacrilamide gel electrophoresis analysis between fresh and frozen semen, either with or without seminal plasma proteins in the cryopreservation medium, revealed that eight protein spots were lost in frozen-thawed sperm. The concentration of one sperm membrane protein spot of low Mr (spot 2) was higher (<em>p </em>&lt; 0.05) in proteinadded frozen sperm. Correlations found between certain protein spots sperm motility and viability suggests that these proteins could play important roles in the maintenance of sperm integrity and functionality. In conclusion, the addition of seminal plasma proteins to freezing extender improved frozen-thawed ram sperm integrity quality and cryopreservation of ram semen produced variations in the sperm membrane protein composition. </p>

Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading

2010 ◽  
Vol 110 (6) ◽  
pp. 1215-1224 ◽  
Author(s):  
Davide Basco ◽  
Grazia Paola Nicchia ◽  
Jean-François Desaphy ◽  
Diana Conte Camerino ◽  
Antonio Frigeri ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Soleus Muscle ◽  
Hindlimb Unloading ◽  
Two Dimensional ◽  
Sds Page ◽  
Rat Soleus Muscle ◽  
Protein Alterations ◽  
Blue Native

Synthesis of a hemifluorinated amphiphile designed for self-assembly and two-dimensional crystallization of membrane protein

2008 ◽  
Vol 49 (14) ◽  
pp. 2247-2250 ◽  
Author(s):  
Julien Dauvergne ◽  
Ange Polidori ◽  
Catherine Vénien-Bryan ◽  
Bernard Pucci
Keyword(s):  
Membrane Protein ◽  
Self Assembly ◽  
Two Dimensional

Oligomerization of polytopic α-helical membrane proteins: causes and consequences

2012 ◽  
Vol 393 (11) ◽  
pp. 1215-1230 ◽  
Author(s):  
Florian Cymer ◽  
Dirk Schneider
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Present Article ◽  
Experimental Information ◽  
Protein Oligomerization ◽  
Open Questions ◽  
Specific Activities ◽  
Physiological Impact ◽  
And Control

Abstract Several polytopic α-helical membrane-integrated proteins appear to be organized in higher-ordered oligomeric complexes. While many aspects are still enigmatic, in recent years, the physiological impact of membrane protein oligomerization has been analyzed to some extent. In the present article, oligomerization of structurally well-defined membrane proteins is discussed. The available experimental information indicates the causes and physiological consequences of membrane protein oligomerization, including stabilization, cooperative functions, and control of specific activities. Based on the currently available observations, we aim to derive some general principles and discuss open questions.

scholarly journals Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

1992 ◽  
Vol 116 (2) ◽  
pp. 339-348 ◽  
Author(s):  
J M Wahlberg ◽  
H Garoff
Keyword(s):  
Membrane Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Quaternary Structure ◽  
Low Ph ◽  
Host Cells ◽  
Protein Oligomerization ◽  
Protein Subunit ◽  
New Host ◽  
E1 Protein

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.

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