title: Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes.
abstract: Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these sources of error can interfere with the method of determining the reversible equilibrium thermodynamic stability of proteins using titrations of chemical denaturants. Here, we present methods to manage light scattering by liposomes for tryptophan emission experiments and to properly monitor tryptophan spectra as a function of protein conformation. Our methods are tailored to the titrations of membrane proteins using common chemical denaturants. One of our recommendations is to collect and analyze the right-angle light scattering peak that occurs around the excitation wavelength in a fluorescence experiment. Another recommendation is to use only those tryptophan spectral parameters that are linearly proportional to the protein conformational population. We show that other commonly used spectral parameters lead to errors in protein stability measurements.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: C Preston
authors.initials: CP
authors.lastname: Moon
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
mesh_terms: D001419:Bacteria / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D008565:Membrane Proteins; D014364:Tryptophan