Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A: Characterization by 1H nuclear magnetic resonance |
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Authors: | Roger G Biringer Anthony L Fink |
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Institution: | Division of Natural Sciences, University of California Santa Cruz, CA 95064, U.S.A. |
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Abstract: | The thermal unfolding of ribonuclease A has been studied in solutions of 25, 35 and 50% methanol (), using 360 MHz proton magnetic resonance spectroscopy. Several observations indicate that the native structure of the protein in methanol cryosolvents is very similar to that in aqueous solution. A detailed analysis of the unfolding process has been made using the C-2 protons of the imidazole side-chains of the four histidine residues. As denaturation proceeds new resonances appear, whose chemical shifts correspond to neither native nor fully unfolded species. These have been assigned to particular His residues by selective deuteration studies. The thermal denaturation transitions reveal a multiphasic process in each of the solvents, and become less co-operative with increasing concentrations of methanol. The denaturation is fully reversible with no evidence of hysteresis.The new resonances that appear during the unfolding process are attributed to partially folded species, which are stabilized by the presence of the relatively hydrophobic methanol. Based on the temperature dependence of the chemical shifts and the relative areas of the various resonances, a detailed sequence of events has been proposed to describe the unfolding process. Key features include the initial general loosening of the two domains, the subsequent movement of the upper S-peptide region (residues 13 to 25) away from the main body of the protein, followed by partial separation of the sheet structure and full exposure of the N-terminal helix, leading to complete separation of the “winged domains”, and ultimately the loss of the residual sheet and helix structure. |
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Keywords: | n m r nuclear magnetic resonance DSS sodium-2 2-dimethyl-2-silapentane-5-sulfonate p p m parts per million u v ultraviolet light |
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