Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence.
Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.
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Transfusion and Cryopreservation Program of the Henry Jackson Foundation for the Advancement of Military Medicine at the Naval Medical Research Center, 8901 Wisconsin Avenue, Bethesda, MD 20889, USA.
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D000818:Animals; D002417:Cattle; D005656:Fungal Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry; D008962:Models, Theoretical; D010735:Phosphoglycerate Kinase / Q000737:chemistry; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
D005656:Fungal Proteins; D007782:Lactoglobulins; D010735:Phosphoglycerate Kinase