Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy.
Fourier transform infrared (FTIR) amide I spectroscopy has not been widely used as a method to study protein folding. Some thorough studies of thermal unfolding have been carried out; however, protein unfolding in the presence of the widely used denaturants guanidine hydrochloride and urea has only recently been reported. Guanidine hydrochloride and urea both absorb strongly in the amide I region, as does H2O. Here, we have used deuterated 13C-urea as the chemical denaturant and monitored the unfolding transition with deuterium-exchanged staphylococcal nuclease (SNase) in D2O. These conditions circumvent all subtraction difficulties as the absorption bands of D2O and denaturant are shifted out of the amide I' region [Fabian, H., and Manstch, H. H. (1995) Biochemistry 34, 13651-13655]. A very reproducible unfolding transition is obtained for SNase. 13C-Urea-induced unfolding of SNase was found not only to be comparable to previous FTIR thermal unfolding data but also to have a denatured-state spectrum similar to those of other thermally denatured proteins. The unfolding is approximately two-state. The infrared spectra in the denatured state show evidence of some residual beta-sheet structure as well as other band components not attributable to random structure.
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Department of Chemistry, University of Denver, Colorado 80208-2436, USA.
Bowler
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D002942:Circular Dichroism; D017666:Deuterium Oxide; D008836:Micrococcal Nuclease / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014508:Urea / Q000378:metabolism*; D014867:Water
D014867:Water; D014508:Urea; D008836:Micrococcal Nuclease; D017666:Deuterium Oxide