Biomedical subjects
Doug Barrick
Publications and source records attributed to Doug Barrick.
Measuring the stability of partly folded proteins using TMAO.
Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.
Relationships between the temperature dependence of solvent denaturation and the denaturant dependence of protein stability curves.
We have used a simple binding model to consider how the thermodynamics of denaturant-protein interactions might influence the shape of protein stability curves (free energy change as a function of temperature), and how these effects translate into a temperature dependence of the apparent m-value (sensitivity of unfolding free energy to denaturant). We find that for an exothermic binding reaction with no binding heat capacity increment, increasing denaturant concentrations produces an apparent increase in curvature in the protein stability curve, giving rise to an increase in the heat capacity increment of unfolding. Similar increases are seen if the binding heat capacity increment is taken as positive. However, for a negative binding heat capacity increment, increasing denaturant concentrations decreases the curvature of the stability curve, giving rise to a decrease in the heat capacity of unfolding. At very high denaturant concentrations (above which the heat capacity of denaturation becomes negative) the stability curve becomes dimpled, showing two separate maxima rather than one. These three models result in very different temperature dependencies of apparent m-values. For urea-induced unfolding of the ankyrin-domain of the Drosophila Notch protein, we find a dependence of experimental m-values on temperature that is similar to that produced by a negative binding heat capacity increment. This temperature dependence is consistent with the observed decrease in heat capacity of unfolding as denaturant is added.
Limits of cooperativity in a structurally modular protein: response of the Notch ankyrin domain to analogous alanine substitutions in each repeat.
To determine the limits of cooperativity in a structurally modular protein, we characterized the structure and stability of glycine variants of the ankyrin repeat domain from the Drosophila melangaster Notch receptor. The substitutions are of analogous alanine residues to glycine in each repeat, and allow the same perturbation to be examined at different positions in the protein. The ankyrin domain is insensitive to substitution in repeat one, suggesting that the first repeat is not fully-folded. Glycine substitutions in repeat two through seven are strongly destabilizing, but the variants retain their overall secondary and tertiary structures. Spectroscopic and calorimetric data are consistent with two-state unfolding transitions for the repeat-two through repeat-five glycine variants, and for the wild-type protein. These data indicate that, despite its modular structure, the Notch ankyrin domain unfolds as a cooperative unit consisting of the six C-terminal repeats, and that this cooperativity is maintained in the presence of severely destabilizing substitutions in the N-terminal and central repeats. In contrast, glycine substitution in repeat six leads to a multi-state unfolding transition, suggesting that the coupling that gives rise to long-range cooperativity in the wild-type protein may have a weak link in the C-terminal region. Such behavior is captured by a simple statistical thermodynamic model in which an unstable C-terminal region is coupled to a stable N-terminal region through a strongly stabilizing interface.
Irreversible assembly of membrane fusion machines.
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