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

Abstract

Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding. These studies uniquely advance our understanding of both the energetics and kinetics of protein folding. Equilibrium studies provide detailed maps of local stabilities, access to energy landscapes, insights into cooperativity, determination of nearest-neighbor interaction parameters using statistical thermodynamics, relationships between consensus sequences and repeat-protein stability. Kinetic studies provide insight into the influence of short-range topology on folding rates, the degree to which folding proceeds by parallel (versus localized) pathways, and the factors that select among multiple potential pathways. The recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings.

Figure 1. Comparison of globular and repeat protein structures

(A) A typical globular protein used in folding studies (RNase A, top; 7RSA.pdb) is compared to (B) a naturally occurring repeat protein (the Notch ankyrin domain, middle; 1OT8.pdb chain A) and to (C) a consensus repeat protein (bottom; 2FO7.pdb). Left: ribbon diagrams (prepared with MacPyMOL [107]) coloring different secondary structure elements (top) and repeats (center, bottom). Middle: contact maps, emphasizing the lack of long-range contacts in repeat proteins (center, bottom), and regular patterns of tertiary structure in different regions of repeat proteins. Right: analogy of globular and repeat proteins to assemblages of fresh fruit. Although the usual fruits of the familiar metaphorical comparison are apples and oranges, some elements of secondary structure (and whole repeat units) are elongated and can be better represented by bananas.

Figure 2. Structure of various repeat proteins

(A) the ankyrin repeats of the Notch receptor, 1O8T.pdb; (B) consensus tetratricorepeats, 2F07.pdb; (C) heat repeats; 1UPK.pdb; (D) internalin-B leucine-rich repeats, 1H62.pdb; (E) hexapeptide repeats, 1J2Z.pdb. Left: overall architecture of single repeats of some of the most common linear repeat proteins. α-helices are red, β-strands are yellow, PPII structure is green, and tight turns are blue. Center: linear arrays of these the same repeats, with adjacent repeats colored from red to purple (N to C). Right: surface representation of adjacent repeats, showing contiguous packing over the entire domain.

Figure 3. Equilibrium two-state and multistate unfolding of repeat proteins

(A) Urea-induced unfolding of the Notch ankyrin domain, monitored by tryptophan fluorescence (circles) and CD (x’s) in the α-helical region. Data are converted to fraction folded to illustrate the coincidence of these two probes. The ribbon model shows the relative positions of the helices and the single tryptophan. Data adapted from [52]. (B) Guanidinium chloride-induced unfolding of pertactin, monitored by tryptophan fluorescence. The unfolding transition shows clear multistate reaction in which an intermediate is formed at ~1.5 M guanidinium chloride. Adapted with permission from [55].

Figure 4. Experimentally determined energy landscapes for helical repeat proteins

(A) Reaction scheme for the Notch ankyrin domain showing transitions between nearest-neighbor conformations on the landscape. (B) Energy landscape of the Notch ankyrin domain. The energies of conformations with blocks of contiguous folded repeats, colored according to free energies, are shown as a function of the number of folded repeats and the location of partly folded structure (as in panel A). (C) Energy landscape for an eight helix consensus TPR construct, using the Ising analysis of Kajander ([42]; see Table 2 for energies). The landscapes in B and C are plotted on the same energy scale, and in both cases the fully folded states (right-most tier) are set to zero energy.