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, 2H218O, glycerol and sucrose29. H218O has the same density as 2H2O and may be a better choice if deuteration of exchangeable protons in the protein is not desired. In this procedure, the density of 3-(N,N-dimethylmyristylammonio)-propanesulfonate (C14SB) detergent in 50 mM Tris pH 7.3, 100 mM NaCl will be matched with 2H2O. As an initial guess the following concentrations of 2H2O will be used: 10, 30, and 50% v/v.
1.1. Sample preparation
<ol>
	<li>Prepare the following stock solutions and filter sterilize through a 0.2 &#956;m syringe filter: 50 mL 500 mM Tris pH 7.3 and 1 M NaCl (10X buffer solution); 1 mL 250 mM C14SB (50X detergent solution).</li>
	<li>Prepare 200 &#956;l of sample solution by mixing 20 &#956;l 10X buffer solution, 4 &#956;l 50X detergent solution, 20 &#956;l 2H2O (99.9%), and 156 &#956;l deionized H2O. Prepare also 200 &#956;l of reference solution by mixing 20 &#956;l 10X buffer solution, 20 &#956;l 2H2O (99.9%), and 160 &#956;l deionized H2O.</li>
	<li>Repeat step 1.1.2 for the other 2H2O concentrations, i.e. 30% and 50%, adjusting the 2H2O and H2O amounts appropriately.</li>
</ol>
1.2. Assembly of 6-channel AUC cells and sample loading into the cells.
Note: There are two types of AUC cell depending on the sample loading method. Cells without external fill has to be loaded prior to sealing the cell, whereas external-fill cells can be loaded after the cells are sealed. Assembly of an external-fill AUC cell has been described previously3. In this protocol, the assembly of a 6-channel AUC cell without external fill is described. The main difference is that it has screw rings on both sides which need to be tightened separately, and it doesn&#8217;t need housing plugs (Fig. 1). The difference in assembly steps are highlighted below.
<img alt="Figure 1" src="/files/ftp_upload/52404/52404fig1.jpg" /> 
Figure 1.&#160;Exploded view of a 6-channel AUC cell without external fill. This figure has been modified from Beckman Coulter An-50 Ti and An-60 Ti Analytical Rotor, Cells, and Counterbalance user manual.
<ol>
	<li>Prepare two window assemblies for each AUC cell with sapphire window instead of quartz window (Fig. 2). Place the window gasket into the window holder. Slightly bend the window liner and place it into the window holder such that the gap is formed opposite to the window holder keyway. Place the sapphire window inside the window liner, aligning the mark with the window holder keyway. 
	Note: The quartz window is compressible and thus will produce more light refraction at high speed 28, 30. Therefore for interference measurements above 30000 rpm, such as in this density matching experiment, sapphire windows are used. A sapphire window is heavier than quartz window and has an &#8220;X&#8221; etched onto its side.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52404/52404fig2.jpg" /> 
Figure 2.&#160;Exploded view of the window assembly. This figure has been modified from Beckman Coulter An-50 Ti and An-60 Ti Analytical Rotor, Cells, and Counterbalance user manual
<ol>
	<li>Place the cell housing with the part number upside-down. With the keyways aligned with the housing key, slide into the cell housing firstly a 6-sector centerpiece with beveled side down, followed by one window assembly with the window facing down (Fig. 1, left).</li>
	<li>Lightly coat the screw ring threads and screw ring washer with spinkote. Place a screw ring washer on top of the window assembly. Install the screw ring into the window housing with the word &#8220;OUT&#8221; facing outside. Hand-tighten the screw ring by using the cell aligning tool.</li>
	<li>Using the torque wrench, tighten the screw ring to only 60 inch-pounds.</li>
	<li>Place the cell with the part number upright and positioned at 12-noon. Load 120 &#956;l reference into the left rows and 110 &#956;l sample into the right rows. Ensure that each sample and reference is correctly paired. 
	Note: Exact sample volume is not critical, but the reference needs to have slightly more volume than the sample (5-10 &#956;l) so that the sample meniscus will be distinct.</li>
	<li>Carefully slide into the cell housing one window assembly with the window facing down (Fig. 1, right). Take care not to disturb the cell excessively and spill the contents.</li>
	<li>Repeat step 1.2.3 and 1.2.4, tightening the second screw ring to 120 inch-pounds. Invert the cell and re-tighten the first screw ring to 120 inch-pounds.</li>
	<li>Load the cells into the rotor, install the rotor into the centrifuge and install the monochromator according to the manufacturer&#8217;s instructions 28. 
	Note: Details on this step can also be found in this reference 3.</li>
</ol>
1.3. Setting up interference measurement
<ol>
	<li>Start up the user interface software for the AUC instrument and perform laser setup and radial calibration for each cell at 3000 rpm, according to the manufacturer&#8217;s instructions, which will be briefly summarized in the following step.</li>
	<li>1.3.1.1 After sufficient vacuum has been reached (&#60; 100 microns), run the centrifuge at 3000 rpm. Preview the interference pattern in the user interface software and adjust the laser parameters to obtain the highest contrast.</li>
	<li>Create a new set up file (File | New File) specifying &#8220;Equilibrium&#8221; and &#8220;Interference&#8221; measurement. Set up a sedimentation equilibrium method (&#8220;Method&#8221; button) to run at 45000 rpm or the highest speed anticipated for protein samples, whichever is higher, with run temperature at 20&#176;C and collect 1 scan every 15 minutes. Monitor equilibrium progress by opening the data files in HeteroAnalysis and selecting &#8220;Match&#8221; function after at least 12 hours (approximately overnight, Fig. 3). 
	Note: a similar function is also available in SEDFIT (Options | Loading Options | Test Approach to Equilibrium).</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52404/52404fig3.jpg" /> 
Figure 3.&#160;Result from HeteroAnalysis Match function. The Match function can be used to monitor equilibrium progress by comparing RMSD between successive scans and the last scan. This example shows attainment of equilibrium after 8 hr as indicated by RMSD values asymptotic to X-axis.
1.4. Data analysis
<ol>
	<li>For each set of samples, plot the slope of the radial distribution profile against the 2H2O concentration. 
	Note: The distribution will be a very shallow exponential that approaches linearity. X-axis intercept corresponds to the matching 2H2O concentration.</li>
	<li>For more accurate results, perform the experiment in several replicates. Alternatively, repeat the experiment with a narrower range of 2H2O concentration.</li>
</ol>
2. Sedimentation equilibrium of SH in C14SB micelles
2.1. Run parameters
<ol>
	<li>Calculate buffer density and viscosity, protein partial specific volume and centrifugation speed by using SEDNTERP. To calculate buffer density and viscosity, select Compute in &#8220;Buffer Data Select&#8221; section and enter the buffer components accordingly, including the D2O concentration.</li>
	<li>2.1.1.1 To calculate protein partial specific volume, select Compute in &#8220;V-bar&#8221; section and enter the protein amino acid sequence. Specify the highest expected oligomeric size in &#8220;Make an oligomer from this monomer: N=&#8221; field, in this case N = 5. Calculate the speed by entering values in the RPM field on the main window until &#963; &#8776; 1; this is a rule of thumb to ensure a good exponential shape of the radial distribution profile25. 
	Note: The values calculated for this experiment was as follows: &#961; = 1.03839 g/ml, &#951; = 1.0267 cP = 0.7569 ml/g, &#969;1 = 16000 rpm.</li>
	<li>Calculate subsequent speeds to follow to ensure sufficient difference between the distribution profile at one speed and the next25. 
	Note: this can also be done from the function &#34;Estimate equilibrium rotor speeds function&#34; in SEDFIT, which takes into account the solution column (filling volume).</li>
</ol>
2.2. Sample preparations
<ol>
	<li>Prepare 1 ml reference solution with 5 mM C14SB and 32.3% 2H2O as determined from density matching experiment (section 1), by mixing 100 &#956;l 10X buffer solution (step 1.1.1), 20 &#956;l 50X detergent solution (step 1.1.1), 323 &#956;l 2H2O (99.9%) and 527 &#956;l deionized H2O.</li>
	<li>Dissolve lyophilized, HPLC-purified SH peptides (expression and purification described previously31) in appropriate solvent such as methanol or 50% v/v aqueous acetonitrile. Measure A280 of the dissolved peptides in a microlitre-scale UV/Vis spectrophotometer and aliquot for three samples to give A280, 12mm = 0.3, 0.5, and 0.8 (A280, 10mm = 0.25, 0.417, and 0.67) each when diluted to 130 &#956;l. Lyophilize the samples overnight and resuspend in 130 &#956;l reference solution (step 2.2.1) to give the sample solutions. 
	Note: SH protein can be detected from UV/Vis absorbance at 280 nm because it contains Trp and Tyr residues. Proteins without aromatic residues can be detected by tagging them with a suitable chromophore, using a Trp-containing mutant, or by using interference measurements instead of absorbance.</li>
	<li>Follow steps in section 1.2 to assemble a 6-channel AUC cell with quartz windows. Load the highest concentration sample (A280, 12mm = 0.8) in the channel nearest to the rotor center and lowest concentration sample (A280, 12mm = 0.3) furthest from the rotor center.</li>
</ol>
2.3. Setting up absorbance measurements
<ol>
	<li>Create a new set up file (File | New File) specifying &#8220;Equilibrium&#8221; and &#8220;Absorbance&#8221; measurement. Specify 280 nm as the detector wavelength.</li>
	<li>Perform radial calibration at 3000 rpm by checking &#8220;Radial calibration before first scan&#8221; in Scan Options, specifying data collection at low resolution, e.g. with Radial step size = 0.01 cm, Replicates = 3 (low resolution, fast), and executing a Single Scan. After the scan is completed, uncheck the option.</li>
	<li>Set up a sedimentation equilibrium method (&#8220;Method&#8221; button) to run at the first speed calculated in step 2.1.3, at 20&#176;C, and collect 1 scan every 30 minutes. In each cells&#8217; &#8220;Detail&#8221;, specify data collection at low resolution as in step 2.3.2. Monitor equilibrium progress by opening the data files in HeteroAnalysis and selecting &#8220;Match&#8221; function after at least 18 hours (approximately overnight, Fig. 4). 
	Note: attainment of equilibrium may take substantially longer time for the first speed, whereas subsequent speeds will take less time.</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52404/52404fig4.jpg" /> 
Figure 4.&#160;Results from HeteroAnalysis Match function. The first and second speeds (top left and right) appear to have reached equilibrium, but it is better to wait a few more hours to be sure. In comparison, the third and fourth speeds (bottom left and right) have clearly reached equilibrium in a shorter time. <a href="https://www.jove.com/files/ftp_upload/52404/52404fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Once equilibrium has been reached, collect a single scan at high resolution, e.g. with Radial step size = 0.001 cm, Replicates = 10 (high resolution, slow).</li>
	<li>After the scan is completed, repeat step 2.3.3 and 2.3.4 for the next speed.</li>
	<li>Optionally, when the time required to reach equilibrium for each speed is known (calculated or from experience), set up the sedimentation equilibrium method scan to include all the speeds calculated in step 2.1.3 and to collect 1 scan after the equilibration time for each speed. In this case, specify high resolution data collection in each cells&#8217; &#8220;Detail&#8221;.</li>
</ol>
2.4. Data analysis in SEDFIT and SEDPHAT
Note: For further details and considerations in data analysis reader is referred to the following website: www.analyticalultracentrifugation.com.
<ol>
	<li>Open high resolution scans in SEDFIT (Data | load sedimentation equilibrium data) and split the data into 3 channels (these correspond to different concentrations for each sample; Options | Loading Options |Save 6-channel Raw Data in 3 Subsets).</li>
	<li>Re-open data files that belong to the same sample and same concentration but different speeds in SEDFIT. Adjust the meniscus (vertical red line), cell bottom (vertical blue line) and fitting limits (vertical green lines), and export the data for use in SEDPHAT (Data | Export Data to SEDPHAT). Input the parameters calculated in step 2.1.1, as well as rotor type and centerpiece type as requested. Repeat this step for every sample and concentration.</li>
	<li>Open all data from the same sample (all concentrations and speeds) in SEDPHAT and fill in Experiment Parameters; an example is shown in Fig. 5. 
	Note: when D2O is added in the buffer, deuteration of exchangeable protons could significantly alter the molecular weight of the protein, especially for water-soluble proteins. Membrane proteins, especially small ones like SH protein, are less affected because membrane-embedded regions are protected from exchange. To correct this, input the &#8220;buffer D mol fraction&#8221;. 
	Note: At this step it is recommended to save the edited dataset separately by selecting menu Data | Copy All Data And Save As New Config.</li>
	<li>Select a Model and fill in Global Parameters for that model. 
	Note: As an example, the &#8220;Monomer-n-Mer Self-Association&#8221; model and its parameters are shown in Fig. 6.</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52404/52404fig5.jpg" /> 
Figure 5.&#160;An example on how to fill in Experimental Parameters.
<img alt="Figure 6" src="/files/ftp_upload/52404/52404fig6.jpg" /> 
Figure 6.&#160;An example on how to fill in Global Parameters for Monomer-n-Mer Self-Association model.
<ol>
	<li>Run a Global Fit by selecting menu Fit | Global Fit and wait until the fit converges. Note down (or take a screenshot of) the fitting results, especially the global reduced chi-square and log Ka values. Extract other data such as fit data and fitting residuals from menu Copy and Display | Display Thermodynamic Information.</li>
	<li>Return to Global Parameters and check M(1) to fit the monomer molecular weight and repeat step 2.4.5. Note down the fitted molecular weight and global reduced chi-square.</li>
	<li>Repeat step 2.4.3 to 2.4.6 for each model to be tested and compare the fit quality of each model by comparing the reduced global chi-square value as well as fitting residuals. 
	Note: Small and random fitting residuals generally indicates a good fit, and the model that fits best would have the smallest global reduced chi-square. The fitted monomer molecular weight and its chi-square value should not differ substantially from that of the fixed (theoretical) molecular weight.</li>
	<li>Calculate the confidence interval for the obtained log Ka by value by first selecting Statistics | Critical chi-square for error surface projections and inputting the desired confidence interval. Next, go to Statistics | Generate 1-dimensional error surface projection and unselect log Ka in Global Parameters dialog to obtain the chi-square values for log Ka. 
	Note: readers are advised to consult the following sources (http://www.analyticalultracentrifugation.com/ sedphat/statistics.htm) for more details on the method 32 as well as illustration of this method 33.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+membrane+protein+molecular+weights+and+association+equilibrium+constants+using+sedimentation+equilibrium+and+sedimentation+velocity.">Determination of membrane protein molecular weights and association equilibrium constants using sedimentation equilibrium and sedimentation velocity.</a> Meth. Cell. Biol. 84, 181-211 (2008).</li><li>Cole, J. L., Lary, J. W., Moody, T. P., Laue, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Ultracentrifugation:+Sedimentation+Velocity+and+Sedimentation+Equilibrium.">Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium.</a> Meth. Cell. Biol. 84, 143-179 (2008).</li><li>Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+membrane+protein+molecular+weight+using+sedimentation+equilibrium+analytical+ultracentrifugation.">Determination of membrane protein molecular weight using sedimentation equilibrium analytical ultracentrifugation.</a> Curr. Protoc. Prot. Sci. 53, 17.12.11-17.12.13 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An-50 Ti and An-60 Ti Analytical Rotor, Cells, and Counterbalance. , (2005).</li><li>Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+membrane+proteins+in+detergent+solution+by+analytical+ultracentrifugation:+Different+methods+for+density+matching.">Studying membrane proteins in detergent solution by analytical ultracentrifugation: Different methods for density matching.</a> Prog. Colloid Polym. Sci. 113, 176-181 (1999).</li><li>Laue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+20.3.">Ch. 20.3.</a> Current Protocols in Protein Science. 20, 20.23.21-20.23.13 (2001).</li><li>Gan, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Small+Hydrophobic+Protein+Of+The+Human+Respiratory+Syncytial+Virus+Forms+Pentameric+Ion+Channels.">The Small Hydrophobic Protein Of The Human Respiratory Syncytial Virus Forms Pentameric Ion Channels.</a> J. Biol. Chem. 287, 24671-24689 (2012).</li><li>Bevington, P. R., Robinson, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Data reduction and error analysis for the physical sciences. 336, (1969).</li><li>Schuck, P., Radu, C. G., Ward, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sedimentation+equilibrium+analysis+of+recombinant+mouse+FcRn+with+murine+IgG1.">Sedimentation equilibrium analysis of recombinant mouse FcRn with murine IgG1.</a> Molecular Immunology. 36, 1117-1125 (1999).</li><li>Gan, S. W., Vararattanavech, A., Nordin, N., Eshaghi, S., Torres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cost-effective+method+for+simultaneous+homo-oligomeric+size+determination+and+monodispersity+conditions+for+membrane+proteins.">A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins.</a> Anal. Biochem. 416, 100-106 (2011).</li><li>Montserret, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+structure+and+ion+channel+activity+of+the+p7+protein+from+hepatitis+C+virus).">NMR structure and ion channel activity of the p7 protein from hepatitis C virus).</a> J. Biol. Chem. 285, 31446-31461 (2010).</li><li>Stouffer, A. L., DeGrado, W. F., Lear, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Ultracentrifugation+Studies+of+the+Influenza+M2+Homotetramerization+Equilibrium+in+Detergent+Solutions.">Analytical Ultracentrifugation Studies of the Influenza M2 Homotetramerization Equilibrium in Detergent Solutions.</a> Progr Colloid Polym Sci. 131, 108-115 (2006).</li><li>Sorkin, A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+and+endocytosis:+Close+encounters+of+many+kinds.">Signal transduction and endocytosis: Close encounters of many kinds.</a> Nat. Rev. Mol. Cell Biol. 3, 600-614 (2002).</li><li>Gan, S. W., Ng, L., Lin, X., Gong, X., Torres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+ion+channel+activity+of+the+human+respiratory+syncytial+virus+(hRSV)+small+hydrophobic+protein+transmembrane+domain.">Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain.</a> Protein science : a publication of the Protein Society. 17, 813-820 (2008).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>3-(N,N-dimethylmyristylammonio)propanesulfonate</td><td>Sigma</td><td>T0807</td><td></td></tr><tr><td>Deuterium oxide 99.8%</td><td>Cambridge Isotope</td><td>DLM-4-99.8</td><td></td></tr><tr><td>An-50 Ti Rotor, Analytical, 8-Place</td><td>Beckman Coulter</td><td>363782</td><td></td></tr><tr><td>An-60 Ti Rotor, Analytical, 4-Place</td><td>Beckman Coulter</td><td>361964</td><td></td></tr><tr><td>Cell housing</td><td>Beckman Coulter</td><td>334784</td><td></td></tr><tr><td>12 mm six-channel centerpiece, epon charcoal-filled</td><td>Beckman Coulter</td><td>331376</td><td></td></tr><tr><td>Window holder</td><td>Beckman Coulter</td><td>305037</td><td></td></tr><tr><td>Window gasket</td><td>Beckman Coulter</td><td>327021</td><td></td></tr><tr><td>Window liner</td><td>Beckman Coulter</td><td>362329</td><td></td></tr><tr><td>Sapphire window</td><td>Beckman Coulter</td><td>307177</td><td></td></tr><tr><td>Quartz window</td><td>Beckman Coulter</td><td>301730</td><td></td></tr><tr><td>Screw-ring washer</td><td>Beckman Coulter</td><td>362328</td><td></td></tr><tr><td>Screw ring</td><td>Beckman Coulter</td><td>301922</td><td></td></tr><tr><td>Spinkote</td><td>Beckman Coulter</td><td>306812</td><td></td></tr><tr><td>Torque stand assembly</td><td>Beckman Coulter</td><td>361318</td><td></td></tr><tr><td>Counterbalance</td><td>Beckman Coulter</td><td>360219</td><td></td></tr><tr><td>Cell alignment tool</td><td>Beckman Coulter</td><td>362340</td><td></td></tr><tr><td>SEDNTERP</td><td></td><td></td><td>http://bitcwiki.sr.unh.edu/index.php/Main_Page</td></tr><tr><td>HeteroAnalysis&amp;nbsp;</td><td></td><td></td><td>http://www.biotech.uconn.edu/auf/?i=aufftp</td></tr><tr><td>SEDFIT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .com/sedfit.htm</td></tr><tr><td>SEDPHAT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .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 Views.  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).

Video: Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bioengineering

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

Genetics

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Summary

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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR