Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease.
We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.
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Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada.
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D000143:Acids / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014465:Ultrasonics; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000378:metabolism
D000143:Acids; D012996:Solutions; D014867:Water; D006859:Hydrogen; D014508:Urea; D008836:Micrococcal Nuclease