Conformational flexibility of avidin: the influence of biotin binding

Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.     

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors

To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the Tm value. Rank ordering the affinity and predictions of the dissociation constant of compounds is obtained by measuring the increase in Tm for different protein-inhibitor complexes. This is the first time an extensive characterization of the TdCD method has been described for characterizing lead inhibitors in a drug discovery mode. The method has several favorable properties. Using the new six-cell Peltier temperature controller for the Jasco 810 spectropolarimeter, one can determine the affinity of 12-18 compounds per day. The method also requires only 20-40 microg protein per sample and can be used to estimate the affinity of compounds with dissociation constants of picomolar to micromolar. An important property of the method for lead discovery is that dissociation constants of approximately 5 microM can be estimated from a single experiment using a low concentration of compound such as 20 microM, which is generally low enough for most small molecules to be soluble for testing. In addition, the method does not require labeling the compound or protein. Although other methods such as isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of binding, ITC requires 1-2 mg protein per sample, cannot readily determine binding constants below nanomolar values, is most versatile with soluble compounds, and has a throughput of two to three experiments per day. The ITC method is not usually used in a high-throughput drug discovery mode; however, using the thermodynamic information from several ITC experiments can make the TdCD method very robust in determining reliable binding constants. Using the kinase inhibitors BMS-250595, purvalanol B, AG-12275, flavopiridol, and several other compounds, it is demonstrated that one can obtain excellent comparisons between the Kd values of binding to CDK2 obtained by TdCD and ITC.     

Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters

We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8-6.9 M), cooperative behavior is observed, which allowed the analysis by the two-state model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of DeltaH(m) on T(m) to the T(m) corresponding to 0 m GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.     

Thermodynamic analysis of denaturant-induced unfolding of HodC69S protein supports a three-state mechanism

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His 6HodC69S, a mutant of 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His 6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.     

Synergistic inhibition of acid-induced protein denaturation by trehalose and NaCl: Thermodynamic and kinetic studies

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.     

Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin

The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40-55 degrees C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (DeltaGu, aq) remains practically same between 40 and 55 degrees C, with a value of 11.8 +/- 0.6 kcal mol(-1) (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.     

Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic Archaea

In this study, the thermodynamic activation parameters of cold-adapted proteins from Archaeaa are described for the first time for the irreversible protein unfolding and ribosome-dependent GTPase activity of elongation factor 2 (EF-2) from the psychrotolerant Methanococcoides burtonii and the thermophilic Methanosarcina thermophila. Thermolability of Methanococcoides burtonii EF-2 was demonstrated by a low activation free-energy of unfolding as a result of low activation-enthalpy. Although structural data for EF-2 are presently limited to protein homology modeling, the observed thermodynamic properties are consistent with a low number of noncovvalent bonds or an altered solvent interaction, causing a loss of entropy during the unfolding process. A physiological concentration of potassium aspartate or potassium glutamate was shown to stabilize both proteins against irreversible denaturation by strengthening noncovalent interactions, as indicated by increased activation enthalpies. The transition state of GTPase activity for Methanococcoides burtonii EF-2 was characterized by a lower activation enthalpy than for Methanosarcina thermophila EF-2. The relative entropy changes could be explained by differential displacement of water molecules during catalysis, resulting in similar activation free energies for both proteins. The presence of solutes was shown to facilitate the breaking of enthalpy-driven interactions and structuring of more water molecules during the reaction. By studying the thermodynamic activation parameters of both GTPase activity and unfolding and examining the effects of intracellular solutes and partner proteins (ribosomes), we were able to identify enthalpic and entropic properties that have evolved in the archaeal EF-2 proteins to enable Methanococcoides burtonii and Methanosarcina thermophila to adapt to their respective thermal environments.     

Predicting the equilibrium protein folding pathway: Structure-based analysis of staphylococcal nuclease

The equilibrium folding pathway of staphylococcal nucleas (SNase) has been approximated using a statistical thermodynamic formalism that utilizes the high-resolution structure of the native state as a template to generate a large ensemble of partially folded states. Close to 400,000 different states ranging from the native to the completely unfolded states were included in the analysis. The probability of each state was estimated using an empirical structural parametrization of the folding energetics. It is shown that this formalism predicts accurately the stability of the protein, the cooperativity of the folding/unfolding transition observed by differential scanning calorimetry (DSC) or urea denaturation and the thermodynamic parameters for unfolding. More importantly, this formalism provides a quantitative account of the experimental hydrogen exchange protection factors measured under native conditions for SNase. These results suggest that the computer-generated distribution of states approximates well the ensemble of conformations existing in solution. Furthermore, this formalism represents the first model capable of quantitatively predicting within a unified framework the probability distribution of states seen under native conditions and its change upon unfolding. © 1997 Wiley-Liss, Inc.     

Unfolding energetics and conformational stability of DLC8 monomer

To understand the rules governing the protein folding process it is essential to study the stability and unfolding of small monomeric proteins. Here, I present the pH dependent thermal unfolding energetics and conformational stability analysis of monomeric Dynein light chain protein (DLC8) in the pH range 3.5-2.0. DLC8 is the smallest and the most conserved light chain among the light chains of the dynein motor assembly. Thermal unfolding of DLC8 monomer is much complex with the presence of transient intermediates, which is in contrast to the notion that small proteins unfold via simple two-state process. The unfolding seems to be more cooperative at lower pH and the temperature of highest conformational stability (T(s)) is found to be maximum (295.7 K) at pH 2.76. Stability curves have been simulated to understand the thermodynamic parameters that govern the shapes of the experimentally obtained curves. Further, an effort has been made to correlate the observed differences in the denaturation energetics with the protein sequence in order to throw light on the structure-folding paradigm of the DLC8 monomer.     

Quantitation of protein-protein interactions by thermal stability shift analysis

Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (～ 100 nM to ～ 100 μM).     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slow β-sheet motions

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of β-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.     

Volume of Hsp90 ligand binding and the unfolding phase diagram as a function of pressure and temperature

Volume changes that accompany protein unfolding and ligand binding are important but largely neglected thermodynamic parameters that may facilitate rational drug design. Here, we determined the volume of lead compound ICPD47 binding to an anticancer target, heat shock protein 90 N-terminal domain, using a pressure shift assay (PressureFluor). The ligand exhibited a stabilizing effect on the protein by increasing its melting pressure and temperature. The Gibbs free energy of unfolding depends on the absence or presence of ligand and has an elliptical shape. Ellipse size increases upon addition of the strongly binding ligand, which stabilizes the protein. The three-dimensional (3D) ellipsoidal surface of the Gibbs free energy of unfolding was calculated with increasing ligand concentrations. The negative volume of ligand binding was relatively large and significantly exceeded the volume of protein unfolding. The pressure shift assay technique could be used to determine the volume changes associated with both protein unfolding as well as ligand binding to protein.     

Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding.

Structural characterization of protein unfolding intermediates [Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318], which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent [5, 5'-dithiobis (2-nitrobenzoic acid)] with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.     

Structural studies of hen egg-white lysozyme dimer: comparison with monomer

A facile method for the formation of covalent bonds between protein molecules is zero length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, we report a cross-linking method for lysozyme and some structural studies as well as catalytic activity assay was performed on lysozyme dimer. The results showed that catalytic activity of lysozyme dimer was the same as monomer. Also, the GdnCl-induced equilibrium unfolding of hen egg-white lysozyme monomer and dimer at pH 2 was studied over a temperature range of 290.7-303.2 K by means of CD spectroscopy. The lack of coincidence between two unfolding curves at 222 and 289 nm in lysozyme dimer was observed, which suggested the existence of intermediate state in unfolding process, while lysozyme monomer showed a single cooperative transition. Thus, the thermodynamic parameters were estimated on the basis of two-state mechanism for lysozyme monomer and three-state one for lysozyme dimer. These results indicated that zero length cross-linking can stabilize the intermediate, so the population of intermediate increased. Our results offer a special opportunity to study the role of intermediates in protein folding mechanisms. In addition thermal unfolding of monomer and dimer in 222 nm was achieved.     

Thermodynamic modulation of light chain amyloid fibril formation

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Differences in the pathways for unfolding and hydrogen exchange among mutants of Escherichia coli alkaline phosphatase

Our initial studies of hydrogen-deuterium (H-D) exchange of tryptophan 109 in Escherichia coli alkaline phosphatase (AP) suggested that significant local unfolding of the protein might occur to allow for the exchange reaction, which is very slow at room temperature (Fischer et al., Biochemistry 39 (2000) 1455-1461). In order to investigate whether the partial unfolding and/or 'breathing' motions leading to H-D exchange were part of the unfolding pathway of the protein we prepared a series of mutants, designed to produce cavities around the exchanging residue, and compared their rates of H-D exchange to their lability (rate of inactivation) in guanidine hydrochloride (Gd:HCl). The complex unfolding kinetics of the mutants in the presence of Gd:HCl showed several components with rates that differed substantially among these proteins, but none of the rates of denaturation induced with Gd:HCl was consistently correlated with the H-D exchange rates. We conclude that the partial opening of the AP structure during the H-D exchange of tryptophan 109, although very slow, is not a rate determining step in the unfolding of this protein.     

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.