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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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.

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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.
<p class="j...

Watch this Scientific Journal Video about Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability at JoVE.com

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).

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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several technological changes to fulfill environmental and economic requirements. These developments have resulted in increasingly severe operating conditions whose suitability for conventional and alternative fuels needs to be addressed. For example, fatty acid methyl esters (FAMEs) introduced as biodiesel are more prone to oxidation and may lead to deposit formation. Although several methods exist to evaluate fuel stability (induction period, peroxides, acids, and insolubles), no technique allows one to monitor the real-time oxidation mechanism and to measure the formation of oxidation intermediates that may lead to deposit formation. In this article, we developed an advanced oxidation procedure (AOP) based on two existing reactors. This procedure allows the simulation of different oxidation conditions and the monitoring of the oxidation progress by the means of macroscopic parameters, such as total acid number (TAN) and advanced analytical methods like gas chromatography coupled to mass spectrometry (GC-MS) and Fourier Transform Infrared - Attenuated Total Reflection (FTIR-ATR). We successfully applied AOP to gain an in-depth understanding of the oxidation kinetics of a model molecule (methyl oleate) and commercial diesel and biodiesel fuels. These developments represent a key strategy for fuel quality monitoring during logistics and on-board utilization.

Oxidation stability of transport fuels has become a concern for future fuel development. This work presents an original methodology developed by IFP Energies Nouvelles for assessing fuel stability using two different reactors. This methodology was successfully applied to gain an in-depth understanding of the oxidation kinetics and pathways of model molecules and commercial fuels.

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

The Effect of Charging and Discharging Lithium Iron Phosphate-graphite Cells at Different Temperatures on Degradation

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. The degradation of the cells is assessed by using 10 charging and discharging temperature permutations ranging from -20 &#176;C to 30 &#176;C. This allows an analysis of the effect of charge and discharge temperatures on aging, and their associations. A total of 100 charge/discharge cycles were carried out. Every 25 cycles a reference cycle was performed to assess the reversible and irreversible capacity degradation. A multi-factor analysis of variance was used, and the experimental results were fitted showing: i) a quadratic relationship between the rate of degradation and the temperature of charge, ii) a linear relationship with the temperature of discharge, and iii) a correlation between the temperature of charge and discharge. It was found that the temperature combination for charging at +30 &#176;C and discharging at -5 &#176;C led to the highest rate of degradation. On the other hand, the cycling in a temperature range from -20 &#176;C to 15 &#176;C (with various combinations of temperatures of charge and discharge), led to a much lower degradation. Additionally, when the temperature of charge is 15 &#176;C, it was found that the degradation rate is nondependent on the temperature of discharge.

This article describes the effect of dissimilar charging/discharging temperatures on the degradation of lithium iron phosphate-graphite pouch cells, aiming at simulating close to real case scenarios. In total, 10 temperature combinations are investigated in the range -20 to 30 &#176;C in order to analyze the impact of temperature on degradation.

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. ...

The Effect of Ultraviolet Radiation on the Chemical Bath Deposition of Bis(thiourea) Cadmium Chloride Crystals and the Subsequent CdS Obtention

In this work, the effects on the preparation of bis(thiourea) cadmium chloride crystals when illuminated with ultraviolet (UV) light at a wavelength of 367 nm using the chemical bath deposition technique are studied comparatively. Two experiments are performed to make a comparison: one without UV light and the other with the aid of UV light. Both experiments are performed under equal conditions, at a temperature of 343 K and with a pH of 3.2. The precursors used are cadmium chloride (CdCl2) and thiourea [CS(NH2)2], which are dissolved in 50 mL of deionized water with an acidic pH. In this experiment, the interaction of electromagnetic radiation is sought at the moment the chemical reaction is carried out. The results demonstrate the existence of an interaction between the crystals and the UV light; the UV light assistance causes crystal growths in an acicular shape. Also, the final product obtained is cadmium sulfide and shows no evident difference when synthesized with or without the use of UV light.

The Effect of Ultraviolet Radiation on the Chemical Bath Deposition of Bisthiourea Cadmium Chloride Crystals and the Subsequent CdS Obtention

This article presents a protocol for the synthesis of bis(thiourea) cadmium chloride crystals by chemical bath deposition. Two experiments are described: one aided by ultraviolet light compared to one without ultraviolet light.

In this work, the effects on the preparation of bis(thiourea) cadmium chloride crystals when illuminated with ultraviolet (UV) light at a wavelength of ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Extraction of Lignin with High &amp;#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Niobium Oxide Films Deposited by Reactive Sputtering: Effect of Oxygen Flow Rate

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition parameters such as gas flow rate that results in changes on composition and thus in the film required properties. In this report, reactive sputtering is used to deposit niobium oxide films. A niobium target is used as metal source and different oxygen flow rates to deposit niobium oxide films. The oxygen flow rate was changed from 3 to 10 sccm. The films deposited under low oxygen flow rates show higher electrical conductivity and provide better perovskite solar cells when used as electron transport layer.

Here, we present a protocol for niobium oxide films deposition by reactive sputtering with different oxygen flow rates for use as an electron transport layer in perovskite solar cells.

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition ...