Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Abstract

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

“…This arrangement can enable sections of repeat proteins to unravel without unfolding the complete structure. We have studied the folding of a number of designed TPR proteins and shown that when partial unfolding occurs, the proteins tend to unravel from the less stable outer repeats first (22)(23)(24). As the N termini of LcrH form the dimeric interface (which our results show is the most thermodynamically stable part of the structure), the unraveling must occur mainly from the C terminus.…”

Fibrous Nanostructures from the Self‐Assembly of Designed Repeat Protein Modules

“…[9,11] Strikingly, as you increase the number of consensus TPR motifs within a protein, large elongated superhelical structures with identical inter-and intra-repeat interactions are produced [11c] (Figure 1). [9] CTPR3 was chosen as 1) it is extremely easy to recombinantly synthesize, [9] 2) it is highly stable (12.0 AE 0.7 kcal mol À1 , at pH 7 [12] ), 3) it is structurally very rigid, [13] and 4) it contains a minimal 3-TPR motif unit that is used ubiquitously throughout Nature as a peptide binding motif. [11b,c] Thus, if an arbitrarily large structure comprised of consecutively arrayed linear repeat units could be produced, it should form helical filaments with a freeenergy stability that is orders of magnitude greater than that of the soluble monomers.…”

“…These monomers were based on the original designed CTPR3 protein (3 consensus TPR motifs of 102 amino acids in total). [9] CTPR3 was chosen as 1) it is extremely easy to recombinantly synthesize, [9] 2) it is highly stable (12.0 AE 0.7 kcal mol À1 , at pH 7 [12] ), 3) it is structurally very rigid, [13] and 4) it contains a minimal 3-TPR motif unit that is used ubiquitously throughout Nature as a peptide binding motif. [14] These features are important, since (1) and (2) provide abundant building blocks that remain folded under a range of conditions, (3) prevents the building blocks from futile intraprotein cyclization, and (4) presents a viable route for future decoration and functionalization.…”

Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks

“…These designs have shown that consensus proteins can be easily lengthened or shortened by the addition or removal of identical repeat motifs. Moreover, increasing the number of stacked repeats generates protein constructs with higher thermodynamic stability that can be modelled and predicted using a relatively simplistic Ising model approximation [13,19,28,[34][35][36][37]. Moreover, increasing the number of stacked repeats generates protein constructs with higher thermodynamic stability that can be modelled and predicted using a relatively simplistic Ising model approximation [13,19,28,[34][35][36][37].…”