Resolving the Challenge of Measuring Ligand Binding to Membrane Proteins by Combining Analytical Ultracentrifugation and Light Scattering Photometry

2012 ◽  
Vol 101 (1) ◽  
pp. 92-101
Author(s):  
J.D. Doran ◽  
A.K. Mohanty ◽  
T. Fox
Keyword(s):  
Light Scattering ◽  
Membrane Proteins ◽  
Ligand Binding ◽  
Analytical Ultracentrifugation

Assessing the Stability of Membrane Proteins to Detect Ligand Binding Using Differential Static Light Scattering

2010 ◽  
Vol 15 (3) ◽  
pp. 314-320 ◽  
Author(s):  
Guillermo A. Senisterra ◽  
Hamed Ghanei ◽  
Galina Khutoreskaya ◽  
Elena Dobrovetsky ◽  
Aled M. Edwards ◽  
...  
Keyword(s):  
Light Scattering ◽  
Membrane Proteins ◽  
Ligand Binding ◽  
3D Structure ◽  
Protein Stabilization ◽  
Static Light Scattering ◽  
Differential Scanning Fluorimetry ◽  
The Stability ◽  
3D Structure Determination

Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg++ channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format.

scholarly journals Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

2016 ◽  
Vol 44 (3) ◽  
pp. 810-823 ◽  
Author(s):  
Rohanah Hussain ◽  
Stephen E. Harding ◽  
Charlotte S. Hughes ◽  
Pikyee Ma ◽  
Simon G. Patching ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Analytical Ultracentrifugation ◽  
Titration Calorimetry ◽  
Bacterial Membrane ◽  
Additional Advantage ◽  
Membrane Sensor ◽  
Sensor Kinases ◽  
High Concentrations

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins–synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs.

scholarly journals The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

1989 ◽  
Vol 109 (4) ◽  
pp. 1537-1547 ◽  
Author(s):  
J H Sinard ◽  
W F Stafford ◽  
T D Pollard
Keyword(s):  
Electron Microscopy ◽  
Light Scattering ◽  
Ionic Strength ◽  
Analytical Ultracentrifugation ◽  
Myosin Ii ◽  
Alkaline Ph ◽  
Predominant Species ◽  
Assembly Mechanism ◽  
Rapid Equilibrium ◽  
Low Ionic Strength

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

scholarly journals Size and Shape of Human Fibrinogen in Solution

1977 ◽  
Author(s):  
V. Hofmann ◽  
P.W. Straub ◽  
T. Binkert ◽  
E. Serrallach ◽  
W. Känzig ◽  
...  
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Analytical Ultracentrifugation ◽  
Rotational Diffusion ◽  
Axial Ratio ◽  
Major Axis ◽  
Fluorescence Depolarization ◽  
Human Fibrinogen ◽  
Size And Shape ◽  
Rotational Diffusion Coefficients

In order to obtain information on size and shape of the fibrinogen molecule in solution the translational diffusion coefficient (DT), the rotational diffusion coefficients (DR⊥ and DR//) and the sedimentation coefficients (S) have been measured on human fibrinogen with a clottaoility above 95%. The methods used were dynamic light scattering, nanosecond fluorescence depolarization and analytical ultracentrifugation. Dynamic light scattering yields DT = 2.0 ± 3% x 10-7 cm2sec–1 at a concentration of 7 mg/ml in 0.15 M Tris-NaCl, pH 7.4. DT is strongly dependent on concentration, being 3.4 ± 10% × 10-7 cm2 sec-1 at 0.1 mg/ml. The rotation along the minor axis as measured with the same method is DR = // 1.5 × 106 sec-1 at 0.1 mg/ml. The rotation along the major axis as measured on fibrinogen labeled with dansylchloride is DR// = 1.5 x106 sec–1. S is also strongly dependent on concentration, being 7.9 S at 0.1 mg/ml, 8.1 S at 1 mg/ml and 6.6 S at 10 mg/ml.These results fit with an elongated molecule having an axial ratio of 7. They are compatible with a MW of 340’000 only for concentrations above 2 mg/ml, while at lower concentrations (0.1 mg/ml) they agree with a MW of approximately half the accepted value.

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