Determinants of Cooperativity in Repeat Protein Folding

Abstract

Repeat proteins have been popular model systems in protein folding research due to their modular architecture. Although each repeat has the same fold, because of sequence variation, the folding energy is not distributed evenly among the repeats. Therefore, it is challenging to quantify the repeat-repeat interactions and the intrinsic folding energies of every repeat. To overcome this difficulty, we designed a consensus ankyrin repeat(CANK), which is brought together to make arrays of identical repeats. By solution NMR, we solved the structure of our three repeat CANK, NRC, and verify that NRC adopts the canonical Ankyrin repeat protein fold. Our SAXS measurements show that CANKs in different length arrays also adopt the same fold. By fitting unfolding transitions of a limited number of constructs to Ising model, we found that the individual repeats have low intrinsic stability, but the interfacial interactions are highly stabilizing. We also extended our Ising formalism to include the temperature dependence of unfolding free energy and we quantified the enthalpic and entropic contribution to intrinsic and interfacial stabilities, as well as, the uneven distribution of heat capacity of unfolding into the intrinsic and interfacial components. Our results suggest that intrinsic folding resembles secondary structure formation(loss of conformational entropy) and interfacial folding involves hydrophobic desolvation(entropy increase and negative heat capacity change upon folding). To separate out the contribution of long range electrostatics to intrinsic and interfacial stability, we measured the salt dependence of CANK stabilities. Our results show that NaCl stabilizes the interfaces and has marginal destabilizing effect on intrinsic stability. Currently we are trying to identify the charge-charge interactions responsible for the high salt dependence of interfacial stability by NMR techniques and single point substitutions.