Folding and dynamics of protein chimeras build from two different folds

点击翻译按钮获取中文摘要

Recombination of protein fragments is a plausible mechanism for the emergence of novel protein folds and the diversification of existing ones. Does Nature select fragments for propagation that minimize misfolding and aggregation in the progeny protein? Also, is the stability and folding of the new protein an equal or asymmetric "sum of parts" or is it independent of the parent proteins? Any fragment specific information encoded during recombination to minimize unproductive pathways in protein folding would likely be lost to mutational drift. However, the study of the folding mechanism and conformational dynamics of synthetic proteins obtained by chimeric fusion of existing fragments could provide clues to how Nature selects against misfolding and aggregation. In this context, we study the folding and dynamics of chimeric proteins created by fusion of subdomain-sized protein fragments. One such chimera is CheYHisF, which was generated from the chemotactic response regulator protein CheY of the flavodoxin-like fold and the histidine biosynthetic pathway protein HisF of the TIM- or (ba)8-barrel fold. CheYHisF unfolds reversibly in guanidinium chloride (GdmCl) via an equilibrium intermediate. Interestingly, this intermediate is not populated when the chimera is denatured in urea. Studying the dynamics of the chimera by backbone amide relaxation measured in the native state via Nuclear Magnetic Resonance spectroscopy identified a single residue as being highly dynamic on the millisecond time scale. This residue is present at the interface of the CheY and HisF fragments and its mutation significantly changed the stability of the native state and the equilibrium intermediate. Here we aim to rationalize the effect of mutations on the stability and folding with respect to redistribution of the hydrophobic clusters inherited in the chimera from its parent proteins. Furthermore, we will study two more related chimeras of CheYHisF probing their conformational dynamics on multiple timescales using NMR spectroscopy to determine location of high flexibility and then employ design rules to improve interfacial packing. We will also study the folding pathways of the three synthetic chimeras and compare it to the parent proteins to reveal similarities and differences among these and extant homologues and to learn about rules for successful recombination of fragments.