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. Between 2 and 5 M GdnHCl, a significant drop in anisotropy was observed, indicating faster rotational mobility of tryptophan residues upon unfolding of GR
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. Lower concentration rates of protein to trypsin were used (100:1, 200:1, and 300:1), but in all these experiments the amount of nondigested EI^sup sc^ in the absence of effectors was insignificant after 1 min of reaction (data not shown)
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. The intensity changes of the various fluorophores upon heating could be classified into three distinct stages
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. These pretransitional conformational changes also resulted in an intermediate with hydrophohic exposure, which could lead to serious aggregation in both chemical denaturants and heat-treatment conditions
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.Sample solutionsSolutions for microscopic observations were prepared by mixing PEG 3000 or PEG 10000 in twice-distilled water until dissolution, adding T4 DNA with further mixing and incubation for 30 min, adding DAPI with further mixing and incubation for 30 min, and finally adding alkali chloride solution and letting the sample solution sit for 1-2 h before observations
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. Bonvin** Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands; and [dagger] Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, Zurich, SwitzerlandSubmitted April 23, 2004, and accepted for publication September 20, 2004
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