Doug Barrick

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.

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 ΔG° for unfolding depends linearly on TMAO concentration, and that the sensitivity of ΔG° 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 ΔG° 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.

The folding of single domain proteins — have we reached a consensus?

Rather than stressing the most recent advances in the field, this review highlights the fundamental topics where disagreement remains and where adequate experimental data are lacking. These topics include properties of the denatured state and the role of residual structure, the nature of the fundamental steps and barriers, the extent of pathway heterogeneity and non-native interactions, recent comparisons between theory and experiment, and finally, dynamical properties of the folding reaction.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch‐mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C‐terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch‐mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 Å resolution. The seventh, C‐terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N‐terminal ankyrin repeat, which contains a 15‐residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch‐effector interactions and map the sites of these interactions.

Studies of the Ankyrin Repeats of the Drosophila melanogaster Notch Receptor. 2. Solution Stability and Cooperativity of Unfolding

To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.

Size and Sequence and the Volume Change of Protein Folding

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chateliers principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the proteins conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

Folding landscapes of ankyrin repeat proteins: experiments meet theory.

Nearly 6% of eukaryotic protein sequences contain ankyrin repeat (AR) domains, which consist of several repeats and often function in binding. AR proteins show highly cooperative folding despite a lack of long-range contacts. Both theory and experiment converge to explain that formation of the interface between elements is more favorable than formation of any individual repeat unit. IkappaBalpha and Notch both undergo partial folding upon binding perhaps influencing the binding free energy. The simple architecture, combined with identification of consensus residues that are important for stability, has enabled systematic perturbation of the energy landscape by single point mutations that affect stability or by addition of consensus repeats. The folding energy landscapes appear highly plastic, with small perturbations re-routing folding pathways.

Protein Folding and Stability Using Denaturants

Measurements of protein folding and thermodynamic stability provide insight into the forces and energetics that determine structure, and can inform on protein domain organization, interdomain interactions, and effects of mutations on structure. This chapter describes methods, theory, and data analysis for the most accessible means to determine the thermodynamics of protein folding: chemical denaturation. Topics include overall features of the folding reaction, advances in instrumentation, optimization of reagent purity, mechanistic models for analysis, and statistical and structural interpretation of fitted thermodynamic parameters. Examples in which stability measurements have provided insight into structure and function will be taken from studies in the authors laboratory on the Notch signaling pathway. It is hoped that this chapter will enable molecular, cell, and structural biologists to make precise measurements of protein stability, and will also provide a strong foundation for biophysics students who wish to undertake experimental studies of protein folding.

The contribution of entropy, enthalpy, and hydrophobic desolvation to cooperativity in repeat-protein folding

Cooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity.

The Leucine-Rich Repeat Domain of Internalin B Folds along a Polarized N-Terminal Pathway

The leucine-rich repeat domain of Internalin B is composed of seven tandem leucine-rich repeats, which each contain a short beta strand connected to a 3(10) helix by a short turn, and an N-terminal alpha-helical capping motif. To determine whether folding proceeds along a single, discrete pathway or multiple, parallel pathways, and to map the structure of the transition state ensemble, we examined the effects of destabilizing substitutions of conserved residues in each repeat. We find that, despite the structural redundancy among the repeats, folding proceeds through an N-terminal transition state ensemble in which the extent of structure formation is biased toward repeats one and two and includes both local and interrepeat interactions. Our results suggest that the N-terminal capping motif serves to polarize the folding pathway by acting as a fast-growing nucleus onto which consecutive repeats fold in the transition state ensemble, and highlight the importance of sequence-specific interactions in pathway selection.