AMPHIPOLS: Applications

Application 4: biophysical approaches
Type of
application		 Rationale Observations Amphipols used References
Size exclusion, immobilized metal and affinity chromatographies, BN-PAGE Purifying and studying MP/ APol complexes. Most chromatographic methods can be resorted to. Reserve ion exchange chromatography to uncharged APols. Avoid fusing tags too close to the transmembrane domain to prevent steric/electrostatic interactions with the column that can reduce the efficiency of binding. SEC tends to overestimate the size of MP/A8-35 complexes. A8-35, SAPols, NAPols,
PC-APols		 Bazzacco et al. (2009, 2012), Champeil et al. (2000), Charvolin et al. (2014), Dahmane et al. (2011,2013), Diab et al. (2007a), Etzkorn et al. (2013), Gohon et al. (2008, 2011), Le Bon et al. (2014a), Martinez et al. (2002), Picard et al. (2006), Prata et al.(2001), Sharma et al. (2012), Sverzhinsky et al. (2014), Tribet et al. (1996, 1997), Zoonens et al. (2007)
Ultracentrifugation Purifying and studying MP/ APol complexes. Sucrose gradient, equilibrium and sedimentation velocity methods can all be used. Separating the contributions of the protein and the APol can be facilitated by the use of deuterated or fluorescent APols. A8-35, DAPol, NAPols,
PC-APols, SAPols		 Althoff et al. (2011), Diab et al. (2007a), Gohon et al. (2008, 2011), Martinez et al. (2002), Prata et al.(2001), Sharma et al. (2012), Sverzhinsky et al. (2014), Tribet et al. (1996, 1997)
Light spectroscopy Analyzing APol-trapped MPs. UV and visible absorption, fluorescence, CD and SRCD spectroscopies can all be used. All current APols interfere with IR absorption spectroscopy in the amide band region, but resonance Raman spectroscopy is accessible. A8-35, NAPols, SAPols See e.g., refs. Dahmane et al. (2013), Gohon et al. (2008), Pocanschi et al. (2006), Polovinkin et al. (2014a), Popot et al. (2011), Tifrea et al. (2011), Zoonens et al. (2007)
Radiation scattering Studying the mass, dimensions and organization of MP/APol complexes. DLS, SAXS and SANS have all been used. Avoiding the presence of small oligomers can be difficult. Separating the contributions of the protein and the APol to SANS signals is greatly helped by contrast-matching the APol using isotopically labeled MP or APol. Plain and deuterated A8-35 Charvolin et al. (2014), Gohon et al. (2008, 2011), Popot et al. (2003), Sharma et al. (2012), Sverzhinsky et al. (2014)
Mass spectrometry Analysis of APol-trapped MPs and proteolytic peptides, identification of bound lipids. MALDI-TOF, ESI–MS and ESI-IMS-MS have all been validated. Subunits and lipids can be detected, and the folded and unfolded states of the proteins distinguished. A8-35 facilitates whole-proteome trypsinolysis and identification of tryptic peptides. A8-35, NAPols Bechara et al. (2012), Catoire et al. (2009), Hopper et al. (2013), Leney et al. (2012), Ning et al. (2013, 2014)
Isoelectrofocusing Improving yields over that in detergent solutions? Requires strictly neutral APols. Preliminary data indicate that the approach is feasible. NAPols Bazzacco (2009)