Elaborating a coiledâ coilâ assembled octahedral protein cage with additional protein domains

De novo design of protein nanoâ cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetryâ based strategy for protein assembly in which short, coiledâ coil sequences mediate the assembly of a protein building block into a cage. The geometry of the cage is specified by the combination of rotational symmetries associated with the coiledâ coil and protein building block. We have used this approach to design wellâ defined octahedral and tetrahedral cages. Here, we show that the cages can be further elaborated and functionalized by the addition of another protein domain to the free end of the coiledâ coil: in this case by fusing maltoseâ binding protein to an octahedral protein cage to produce a structure with a designed molecular weight of ~1.8 MDa. Importantly, the addition of the maltose binding protein domain dramatically improved the efficiency of assembly, resulting in ~ 60â fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiledâ coil motifs as offâ theâ shelf components to design MDaâ sized protein cages to which additional structural or functional elements can be added in a modular manner.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146469/1/pro3497.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146469/2/pro3497_am.pd

Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ Designed Coiled Coils

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of proteinâ protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiledâ coil domains to assemble proteins into higherâ order structures. Here, we demonstrate the assembly of a trimeric enzyme into a wellâ defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiledâ coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiledâ coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.Geometry lesson: A modular approach for assembling proteins into largeâ scale geometric structures was developed in which coiledâ coil domains acted as â twist tiesâ to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/1/cbic201700406_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/2/cbic201700406.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/3/cbic201700406-sup-0001-misc_information.pd

Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Chaperones assist the folding of many proteins in the cell. While the most well studied chaperones use cycles of ATP binding and hydrolysis to assist protein folding, a number of chaperones have been identified that promote protein folding in the absence of highenergy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we have characterized the kinetic mechanism of substrate folding by the small, ATP-independent chaperone, Spy. Spy rapidly associates with its substrate, Immunity protein 7 (Im7), eliminating its potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while remaining bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones can assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while continuously bound to a chaperone

Determining crystal structures through crowdsourcing and coursework

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality

Folding while Bound to Chaperones

Chaperones are important in preventing protein aggregation and aiding protein folding. How chaperones aid protein folding remains a key question in understanding their mechanism. The possibility of proteins folding while bound to chaperones was reintroduced recently with the chaperone Spy, many years after the phenomenon was first reported with the chaperones GroEL and SecB. In this review, we discuss the salient features of folding while bound in the cases for which it has been observed and speculate about its biological importance and possible occurrence in other chaperones

A R T I C L E S Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Chaperones are essential for maintaining protein-folding homeostasis, and they have important roles in the cellular stress response. By binding to aggregation-sensitive folding intermediates, chaperones inhibit aberrant interactions between proteins. The most intensively studied folding chaperones, such as the GroEL-GroES and DnaK systems, facilitate substrate protein folding through ATP-and cofactor-driven conformational changes In contrast to ATP-dependent chaperones, a number of chaperones have been identified that can assist in protein folding in the absence of ATP The mechanism by which Spy and the growing class of ATPindependent chaperones function to refold proteins in the absence of energy cofactors is an unresolved question. We addressed this by determining how Spy affects the folding pathway of Im7, the protein that was used to discover Spy. Im7 has a number of attributes that facilitate its study. It is small and monomeric, and it folds via a wellcharacterized mechanism that involves transition through a partially folded on-pathway intermediate before the native state is reache

Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum,”

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection. chaperone | Leishmania | peroxiredoxi