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The design of completely synthetic proteins from first principles— de novo protein design—is challenging. This is because, despite recent advances in computational protein–structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein–protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg — i.e. , the sequence signature of many helical bundles—the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli . All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design. 

Abstract As glaciers around the world rapidly lose mass, the tight coupling between glaciers and downstream ecosystems is resulting in widespread impacts on global hydrologic and biogeochemical cycling. However, a range of challenges make it difficult to conduct research in glacierized systems, and our knowledge of seasonally changing hydrologic processes and solute sources and signatures is limited. This in turn hampers our ability to make predictions on solute composition and flux. We conducted a broad water sampling campaign in order to understand the present‐day partitioning of water sources and associated solutes in Alaska's Wolverine Glacier watershed. We established a relationship between electrical conductivity and streamflow at the watershed outlet to divide the melt season into four hydroclimatic periods. Across hydroclimatic periods, we observed a shift in nonglacial source waters from snowmelt‐dominated overland and shallow subsurface flow paths to deeper groundwater flow paths. We also observed the shift from a low‐ to high‐efficiency subglacial drainage network and the associated flushing of water stored subglacially with higher solute loads. We used calcium, the dominant dissolved ion, from watershed outlet samples to estimate solute fluxes for each hydroclimatic period across two melt seasons. We found between 40% and 55% of Ca²⁺export occurred during the late season rainy period. This partitioning of the melt season coupled with a characterization of the chemical makeup and magnitude of solute export provides new insight into a rapidly changing watershed and creates a framework to quantify and predict changes to solute fluxes across a melt season.