Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present a kinetic method to determine the concentration of native molecules in protein folding transitions. It is based on the observation that frequently native protein molecules unfold slowly when transferred to unfolding conditions, whereas folding intermediates unfold rapidly. The fraction of native molecules in a folding transition can thus be determined by kinetic unfolding assays in a two-step procedure. Aliquots of the protein are first equilibrated at different concentrations of denaturant and then transferred to constant unfolding conditions to determine the amplitude of unfolding. This amplitude is a direct measure for the concentration of native molecules in the sample. The two-state character of a solvent-induced unfolding transition can thus be examined. When the fractional change of a spectral property in a transition follows the decrease in the concentration of the native molecules, as measured by the unfolding assays, then the presence of intermediates that differ from the unfolded protein in this property can be definitely excluded. This test complements the calorimetric test for intermediates in thermal unfolding transitions. By using this method, we show that the NaCl-induced folding transition of the reduced and carboxymethylated form of a variant of ribonuclease T1 is well described by the two-state approximation. In the unfolding of apo-alpha-lactalbumin, the measured profile for the native protein coincides with the fluorescence-detected transition, but not with the transition that is monitored by amide circular dichroism. This confirms that a partially folded intermediate is present in the folding transition of apo-alpha-lactalbumin.
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PMID:A kinetic method to evaluate the two-state character of solvent-induced protein denaturation. 794 99

Mixed disulfides between glutathione and the reduced forms of disulfide-bonded proteins were generated and characterized to explore their suitability as models of the unfolded state of newly-synthesized secretory proteins. RNase T1 and alpha-lactalbumin were reduced and converted to mixed disulfide derivatives, named GS-RNase T1 and GS-alpha-lactalbumin, in good yield; the molecular masses of the derivatives were confirmed by electrospray mass spectrometry. The intrinsic fluorescence of the derivatives and the binding of the hydrophobic fluorescent dye ANS were characteristic of fully unfolded proteins. Fluorescence studies and enzyme activity data indicated that GS-RNase T1 could be refolded to a nativelike state at NaCl concentrations greater than 1.5 M, as was previously demonstrated for the reduced, carboxymethylated derivative of this protein. The [NaCl]-dependent folding/unfolding equilibrium for GS-RNase T1 was reversible and could be influenced by urea. Fluorescence studies indicated that GS-alpha-lactalbumin showed a [NaCl]-dependent partial shift toward a more nativelike state, which was enhanced by the presence of Ca2+ ions. Both of the GS derivatives stimulated the ATPase activity of BiP, with apparent affinities in the range 0.1-1.0 mM. The results indicate that these GS-S-protein mixed disulfide derivatives are ideal model unfolded proteins that can be used as substrates for detailed studies on secretory protein folding in vitro and on the interactions between unfolded proteins and facilitators of protein folding.
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PMID:Protein-S-S-glutathione mixed disulfides as models of unfolded proteins. 801 32

The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions. Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium. The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors. We used this inhibition to characterize the substrate binding sites of the trigger factors. Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M. genitalium with a KI value of 50 nM. The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues. The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding. The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already. They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain.
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PMID:Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues. 953 90