This work has been funded by the National Research Foundation grant NRF-CRP4-2008-02 (J.T.) and Tier 1 grant RG 51/13.

This protocol is based on the following resources, which are to be referred for more details and special considerations3,25-28.
1. Density matching of detergent micelles with 2H2O
Note: The density of the buffer solution needs to be matched to the density of the detergent micelles. Common density-adjusting agents include 2H2O, H218O, 2H218</sup...

<ol>
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This paper provides an experimental protocol for sample preparation and analysis of oligomerization of a small membrane protein in detergent using equilibrium sedimentation. The protocol described is equally valid &#8211;and simpler- for soluble proteins, as the density matching step is not required. Indeed, the system is constituted by a mixture of detergent and protein. To conduct sedimentation studies, the detergent must be invisible to the gravitational field so that it does not contribute to the particle flotation. ...

Analytical ultracentrifugation1-5 is one of the most important methods to study interactions of macromolecules under physiological conditions, being accessible to both weak and strong interactions. The method is label-free and uses light absorption or interference, and even fluorescence optical systems can be used to access concentration ranges over several orders of magnitude6.
This method is especially useful since most biochemical processes depend on reversible interactions. The stoichiometry and strength of these interactions have to be quantitatively characterized to understand biological processes, and a number of methods exist for this purpose 7,8. However, transient interactions are difficult to study 9.
The choice of a method to characterize macromolecular interactions depends on its static or dynamic nature. In the first case, sedimentation velocity (SV) is used, where the rate of radial transport is measured and complexes are fractionated on the basis of differences in buoyant mass and shape.
In contrast, dynamic associations that are reversible on the time scale of the experiment cannot be physically separated. In this case, self- or hetero-interactions leading to non-covalent interactions are in an equilibrium that depends on the total protein concentration. These dynamic interactions can be studied by both sedimentation equilibrium (SE) and sedimentation velocity (SV) 10. However, the first method is simpler to perform and is described here. In SE, centrifugation is performed at a sufficiently low speed so that an equilibrium is reached between diffusion and sedimentation. At this point, the equilibrium profile of an optical signal (UV-VIS) as a function of radial distance, can be analyzed using pre-set thermodynamic models for associations11.
In the present paper, a sedimentation equilibrium study is presented of the self-association of a viral membrane protein that forms ion channels. Because of its hydrophobicity, the experiment is run in presence of detergent, and in this case the density of solvent has to be matched to that of the detergent. However, the protocol described would identical in the case of a water soluble protein, except that no solvent density matching would be required.
The protein used is encoded in the human respiratory syncytial virus (hRSV), an enveloped pneumovirus in the paramyxoviridae family that causes lower respiratory tract disease in infants, elderly and immunocompromised populations worldwide 12. Up to 64 million reported cases of hRSV infection and 160,000 deaths occur each year.
The hRSV genome transcribes 11 proteins, including the three membrane proteins F, G, and small hydrophobic (SH). SH protein is involved in the pathogenesis of RSV infection. RSV lacking the SH gene (RSV&#916;SH) was viable, caused formation of syncytia and grew as well as the wild-type (WT) virus 13-16. However, RSV&#916;SH virus replicated 10-fold less efficiently than the WT in the upper respiratory tract 15,16. Also, RSV&#916;SH virus was attenuated in in vivo mouse and chimpanzee models 13,17.
The SH protein is a 64 (RSV subgroup A) or 65 (RSV subgroup B) amino acids long type II integral membrane protein that &#160;accumulates mostly at the membranes of the Golgi compartment 18. &#160;SH protein has a single predicted a-helical transmembrane (TM) domain 19 which is highly conserved 20,21. The C- and N-terminal extramembrane domains are oriented lumenally/extracellularly and cytoplasmically, respectively.
Both synthetic TM domain (residues 18-43) and full length SH protein have been shown to form homopentamers in a variety of detergents. The homopentameric form is responsible for channel activity in planar lipid bilayers 22,23. The correct orientation of the TM monomers in the lipid bilayer was first determined using site specific infrared dichroism 23, which showed His-22 to be in a lumenal, close to inter-helical, orientation. The same TM domain orientation was confirmed by NMR studies that reconstructed the pentameric a-helical bundle of the full-length protein in dodecylphosphocholine (DPC) micelles 22. In this &#8216;micelle&#8217; model, a single a- helical TM domain was flanked N-terminally by an a-helix, and C-terminally by an extended b-hairpin. The two protonatable residues of SH protein, His-22 and His-51, are located in the TM domain (lumenally oriented), and at the tip of the extramembrane C-terminal &#946; hairpin (far from the channel pore), respectively. In a bicellar environment, however, the TM &#945;-helix&#160; extends up to His-51, and both His residues are accessible to the lumen of the channel24. The channel structure adopts a &#160;funnel-like architecture 22, where the narrower region (Ser-29 to Cys-45) 22 is lined with hydrophobic side chains (Ile-32, Ile-36, Ile-40 and Leu-44), and Ile-36 defines the narrowest point in the channel lumen. His-22 is located at the largest opening of this funnel, whereas His-51 is at the tip of the smallest opening.
In the present paper, analytical centrifugation in a sedimentation equilibrium mode has been used to determine if His protonation affects the stability of the SH protein pentamer. In this case, SH protein was solubilized in C14-betaine detergent, which has been used previously to show that SH protein forms pentameric oligomers22.

<table border="0" cellpadding="0" cellspacing="0" width="1345">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:448px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">3-(N,N-dimethylmyristylammonio)propanesulfonate</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">T0807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Deuterium oxide 99.8%</td>
			<td style="width:147px;">Cambridge Isotope</td>
			<td style="width:119px;">DLM-4-99.8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">An-50 Ti Rotor, Analytical, 8-Place</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">363782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">An-60 Ti Rotor, Analytical, 4-Place</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">361964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Cell housing</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">334784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">12 mm six-channel centerpiece, epon charcoal-filled</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">331376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window holder</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">305037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window gasket</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">327021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window liner</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">362329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Sapphire window</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">307177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Quartz window</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">301730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Screw-ring washer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">362328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Screw ring</td>
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			<td align="right" style="width:119px;">301922</td>
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			<td height="21" style="height:21px;width:632px;">Spinkote</td>
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			<td height="21" style="height:21px;width:632px;">Torque stand assembly</td>
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			<td align="right" style="width:119px;">361318</td>
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			<td height="21" style="height:21px;width:632px;">Counterbalance</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">360219</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Cell alignment tool</td>
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			<td height="21" style="height:21px;">SEDNTERP</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://bitcwiki.sr.unh.edu/index.php/Main_Page</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HeteroAnalysis&#160;</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.biotech.uconn.edu/auf/?i=aufftp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SEDFIT</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.analyticalultracentrifugation 
			.com/sedfit.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SEDPHAT</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.analyticalultracentrifugation 
			.com/sedphat/default.htm</td>
		</tr>
	</tbody>
</table>

sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

The radial distribution profile of C14SB detergent micelles in 50 mM Tris, 100 mM NaCl pH 7.3 forms a very shallow exponential that could be fitted to a linear model (Figure 7A). The slope of this distribution is inversely correlated to D2O concentration (Figure 7B). The point where the slope is zero, i.e., the matching D2O concentration, was found to be 32.3%.
See Figure 7 Below.
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Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

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10.5K visualizações.  Nanyang Technological University. Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).