Thermodynamic Coupling in a Consensus-Designed Spectrin Repeat Protein

Cooperativity is a central feature to protein folding and is important for protein design. Repeat proteins are good systems for quantifying the thermodynamic basis of cooperativity. Analysis of repeat proteins composed of identical consensus repeats show that repeats strongly drive the folding of their neighbors through extensive tertiary contacts. Here, we use the consensus approach to quantify the cooperativity of folding of spectrin repeat arrays. Spectrin repeats are unique among tandem repeat proteins in that they share an elongated α-helix that spans neighboring repeats. We generate a consensus spectrin repeat sequence and show that this sequence is structured by CD and NMR spectroscopy, and is considerably more stable than extant spectrin repeats. By generating pairs of consensus spectrin repeats, we find tandem repeats to be further stabilized, demonstrating cooperative stabilization by neighboring repeats. Using an Ising model to analyze single- and tandem spectrin repeat unfolding, we find that the consensus stability increase results from intrinsic but not interfacial stabilization. By introducing mutations and insertions at the boundary between consensus repeats, we find that cooperativity is driven primarily by helical propagation; to a lesser extent, helix propagation also stabilizes partly folded states where one of two repeats is unfolded.