Engineered Pathways for Correct Disulfide Bond Oxidation

Correct formation of disulfide bonds is critical for protein folding. We find that cells lacking protein disulfide isomerases (PDIs) can use alternative mechanisms for correct disulfide bond formation. By linking correct disulfide bond formation to antibiotic resistance, we selected mutants that catalyze correct disulfide formation in the absence of DsbC, Escherichia coli's PDI. Most of our mutants massively overproduce the disulfide oxidase DsbA and change its redox status. They enhance DsbA's ability to directly form the correct disulfides by increasing the level of mixed disulfides between DsbA and substrate proteins. One mutant operates via a different mechanism; it contains mutations in DsbB and CpxR that alter the redox environment of the periplasm and increases the level of the chaperone/protease DegP, allowing DsbA to gain disulfide isomerase ability in vivo. Thus, given the proper expression level, redox status, and chaperone assistance, the oxidase DsbA can readily function in vivo to catalyze the folding of proteins with complex disulfide bond connectivities. Our selection reveals versatile strategies for correct disulfide formation in vivo. Remarkably, our evolution of new pathways for correct disulfide bond formation in E. coli mimics eukaryotic PDI, a highly abundant partially reduced protein with chaperone activity. Antioxid. Redox Signal. 14, 2399-2412.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90429/1/ars-2E2010-2E3782.pd

Chaperone OsmY facilitates the biogenesis of a major family of autotransporters

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152790/1/mmi14358.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152790/2/mmi14358_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152790/3/mmi14358-sup-0001-Supinfo.pd

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

Oxidative protein folding in bacteria

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli , DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75150/1/j.1365-2958.2002.02851.x.pd

Computational redesign of thioredoxin is hypersensitive towards minor conformational changes in the backbone template

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations

Using Network Component Analysis to Dissect Regulatory Networks Mediated by Transcription Factors in Yeast

Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA) to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs) perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request

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

The geochemical cycling of reactive chlorine through the marine troposphere

Heterogeneous reactions involving sea‐salt aerosol in the marine troposphere are the major global source for volatile inorganic chlorine. We measured reactant and product species hypothesized to be associated with these chemical transformations as a function of phase, particle size, and altitude over the North Atlantic Ocean during the summer of 1988. Concentrations of HCl were typically less than 1.0 ppbv near the sea surface and decreased with altitude and with distance from the U.S. east coast. Concentrations of Cl volatilized from aerosols were generally equivalent to the corresponding concentrations of HCl and ranged from less than detection limits to 125 nmol m−3 STP. Highest absolute and percentage losses of particulate Cl were typically associated with elevated concentrations of anthropogenic combustion products. Concentrations of product nss SO42− and N03− in coarse aerosol fractions indicate that on average only 38% of measured Cl− deficits could be accounted for by the combined effects of acid‐base desorption and reactions involving nonacidic N gases. We hypothesize a mechanism for the Cl loss initiated by reaction of O3 at sea‐salt aerosol surfaces, generating Cl2 followed by rapid photochemical conversion of Cl2 to HCl via Cl atoms (Cl˙) and eventual recapture of HCl by the aerosol. Simulations with a zero‐dimension (0‐D) photochemical model suggest that oxidation by Cl˙ may be an important tropospheric sink for dimethyl sulfide and hydrocarbons. Under low‐NOx conditions, the rapid cycling of reactive Cl would provide a catalytic loss mechanism for O3, which would possibly explain the low O3 concentrations often observed above the world\u27s oceans

Adaptive Evolution in Zinc Finger Transcription Factors

The majority of human genes are conserved among mammals, but some gene families have undergone extensive expansion in particular lineages. Here, we present an evolutionary analysis of one such gene family, the poly–zinc-finger (poly-ZF) genes. The human genome encodes approximately 700 members of the poly-ZF family of putative transcriptional repressors, many of which have associated KRAB, SCAN, or BTB domains. Analysis of the gene family across the tree of life indicates that the gene family arose from a small ancestral group of eukaryotic zinc-finger transcription factors through many repeated gene duplications accompanied by functional divergence. The ancestral gene family has probably expanded independently in several lineages, including mammals and some fishes. Investigation of adaptive evolution among recent paralogs using dN/dS analysis indicates that a major component of the selective pressure acting on these genes has been positive selection to change their DNA-binding specificity. These results suggest that the poly-ZF genes are a major source of new transcriptional repression activity in humans and other primates