Elucidating the Folding Pathways of Calcium-Binding Proteins

During translation, ribosomes synthesize proteins according to the messenger RNA template. The polypeptide chain, specified by the template, acquires its three dimensional structure either co- or post-translationally in a process termed “folding”. Understanding folding mechanisms is important since protein structure is critical for biological function, and misfolded proteins are correlated with cell stress and disease. Here, we map the folding pathways of two distantly related proteins: Calmodulin, a eukaryotic, calcium-dependent signaling protein, and Calerythrin, a prokaryotic, calcium-buffering protein. These proteins have a highly conserved sequence and structure dictated by their similar calcium-binding function. Both proteins have two domains, and each domain is composed of two “EF-hand” motifs. To probe the folding of these proteins, we utilize a focused laser beam to form an optical trap and exert mechanical force on the molecule, while measuring the molecule's response to force via its change in extension. These single-molecule experiments reveal the folding dynamics at a level of detail not possible by traditional ensemble methods, since we can directly observe folding intermediates and off-pathway states. We find that domain proximity, in this case determined by the length of a bridging helix, impacts folding and unfolding cooperativity even though the proteins share folding motifs. Studying these folding mechanisms allows us to obtain a better understanding of how domains communicate with each other and how tertiary contacts affect protein stability, while prompting further studies to examine their structure-function relationship.