Abstract
Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.
Highlights
Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process
This is unfortunate because observation of both unfolding temperatures is in principle very valuable as it allows the calculation of reliable stability curves of the protein and of the whole set of thermodynamic parameters
We recently showed, using Yfh[1] as a suitable model, that it is possible to use 2D NMR to measure protein stability and get thermodynamic parameters comparable to those obtained by CD13
Summary
Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do provide precious information By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different. The reason why cold denaturation is much less understood than heat transition is that most proteins undergo cold denaturation at temperatures below the water freezing point This is unfortunate because observation of both unfolding temperatures is in principle very valuable as it allows the calculation of reliable stability curves of the protein and of the whole set of thermodynamic parameters. When monitoring the unfolding of a protein by CD spectroscopy, for instance, we observe intensity changes related to the disruption of alpha helices and/or beta-sheets under the influence of physical or chemical agents
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