Studies of the Ankyrin Repeats of the Drosophila melanogaster Notch Receptor. 1. Solution Conformational and Hydrodynamic Properties

Abstract

To gain insight into the structural basis for Notch signaling, and to investigate the relationship between structure and stability in ankyrin repeat proteins, we have examined structural features of polypeptides from the Drosophila melanogaster Notch protein that contain five, six, and a putative seventh ankyrin repeat. Circular dichroism (CD) spectroscopy indicates that Notch ankyrin polypeptides of different length contain a significant amount of alpha-helix, indicating that a folded structure can be maintained despite the loss of individual ankyrin modules. However, the alpha-helical content of the construct with the putative seventh repeat is slightly higher than polypeptides containing fewer repeats, suggesting that the putative seventh repeat may help stabilize other parts of the ankyrin domain. Fluorescence spectroscopy indicates that the single tryptophan in the fifth ankyrin repeat is in a nonpolar environment and is shielded from solvent in all three constructs, although slight differences in spectroscopic properties of the six- and five-repeat constructs are observed, indicating minor structural changes. Near-UV CD indicates that these ankyrin polypeptides contain a significant amount of fixed tertiary structure surrounding their aromatic side chains. Gel filtration chromatography and sedimentation equilibrium studies indicate that these ankyrin repeat polypeptides are monomeric. Sedimentation velocity studies indicate that each polypeptide exhibits significant axial asymmetry, consistent with the elongated structure seen for other for ankyrin repeat proteins. However, the degree of asymmetry is greatest for the construct containing six repeats, suggesting that in the absence of the putative seventh repeat, the sixth repeat is partly unfolded.

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…2 Although this combination of modest sequence identity and small repeat size would be expected to seriously hinder sequence-based searches for single repeats, resulting in a large number of false-positives, tandem repetition in repeat arrays (typically four or more copies) greatly increases confidence in sequence searches. However, since repeat proteins are often embedded in larger, non-repeat sequences, identification of the bona fide ends of repeat proteins (and thus the overall length of the repeat domain) from sequence alone can be problematic, both because of the false-positives and false-negatives (for the latter case, terminal repeats often tend to differ in sequence from the consensus to avoid unfavorable contacts with solvent [28][29][30].…”

“…2 Although this combination of modest sequence identity and small repeat size would be expected to seriously hinder sequence-based searches for single repeats, resulting in a large number of false-positives, tandem repetition in repeat arrays (typically four or more copies) greatly increases confidence in sequence searches. However, since repeat proteins are often embedded in larger, non-repeat sequences, identification of the bona fide ends of repeat proteins (and thus the overall length of the repeat domain) from sequence alone can be problematic, both because of the false-positives and false-negatives (for the latter case, terminal repeats often tend to differ in sequence from the consensus to avoid unfavorable contacts with solvent [28][29][30].In the repeat proteins described here (which include families that are abundant in sequence databases, have known high-resolution structures, and have been used as systems for studying folding), adjacent sequence repeats pack together to form an extended array ( Figure 1, Figure 2). Although minor tilts and rotations about the long-axis of the array produce gradual superhelical trajectories (as elegantly detailed Kobe and Kajava [31]), the overall architecture of the repeat proteins described here can be well-approximated as linear.…”

Mixed osmolytes: The degree to which one osmolyte affects the protein stabilizing ability of another

“…The C-terminal His 6 -tagged Nank4-7* construct used in this study contains four tandem ankyrin repeat sequences and was expressed and purified as described previously (Zweifel and Barrick 2001a;Mello and Barrick 2003). pH and salt sensitivity studies Temperature scans from 5°C to 65°C for the pH and NaCl sensitivity studies were performed using a Jasco J-720 spectropolarimeter at 222 nm with a temperature scan slope of 1°C/min. Nank4-7* was present at 0.18 mg/mL, and a 1-mm cuvette completely filled and sealed was used to avoid concentration of the sample by evaporation at higher temperatures.…”

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

“…Variants of the Notch ankyrin domain were expressed in the E. coli BL21(DE3) cell line and purified as described elsewhere. 45 Urea-induced equilibrium unfolding transitions were determined by CD spectroscopy as described in Ref. 46, and are shown for the DG3* and DG3*/LG7* in Supporting Figure 2(A).…”