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Northampton, MA) by using a protein concentration of 1
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. Thermal denaturations following the ANS fluorescence did not show a native baseline, and a sigmoidal-like decreasing of the fluorescence was observed as the temperature was raised (data not shown)
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. The fluorescence emission was collected using the same objective, separated from the excitation light using a dichroic filter (540 DRLP, Omega Optical, Brattleboro, VT), spatially filtered using a 100-µ pinhole, and separated into donor and acceptor components using a second dichroic filter (630 DRLP, Omega Optical)
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