Human amylin, or islet amyloid polypeptide, is a peptide co-secreted with insulin by the beta cells of the pancreatic islets of Langerhans. The 37-residue, C-terminally amidated human amylin peptide derives from a proprotein that undergoes disulfide bond formation in the endoplasmic reticulum and is then subjected to four enzymatic processing events in the immature secretory granule. Human amylin forms both intracellular and extracellular amyloid deposits in the pancreas of most Type II diabetic subjects, likely reflecting compromised secretory cell function. In addition, amylin processing intermediates, postulated to initiate intracellular amyloidogenesis, have been reported as components of intracellular amyloid in beta cells. We investigated the amyloidogenicity of amylin and its processing intermediates in vitro. Chaotrope-denatured amylin and amylin processing intermediates were subjected to size exclusion chromatography, affording high concentrations of monomeric peptides. NMR studies reveal that human amylin samples helical conformations. Under conditions mimicking the immature secretory granule (37°C, pH 6), amylin forms amyloid aggregates more rapidly than its processing intermediates, and more rapidly than its reduced counterparts. Our studies also show that the amyloidogenicity of amylin and its processing intermediates is negatively correlated with net charge and charge at the C-terminus. Although our conditions may not precisely reflect the environment of amyloidogenesis in vivo, the lower amyloidogenicity of the processing intermediates relative to amylin suggests their presence in intracellular amyloid deposits in the increasingly stressed beta cells of diabetic subjects may be a consequence of general defects in protein homeostasis control known to occur in diabetes.In type II diabetes, an increasing demand for insulin places a substantial stress on the protein secretion system of the beta cells of the islets of Langerhans, which results in cellular dysfunction and, eventually, beta cell death. As the population of beta cells continues to diminish, the stress on the remaining cells increases as they struggle to produce the insulin † We thank the NIH (DK46335 and AG18917), The Skaggs Institute of Chemical Biology, and the Lita Annenberg Hazen Foundation for financial support. *Corresponding author: jkelly@scripps.edu, phone: +1-858-784-9880, fax: +1-858-784-9610. SUPPORTING INFORMATION AVAILABLERepresentative analytical ultracentrifugation data; NMR spectra, NMR resonance assignments and a table of resonances; as well as representative amyloidogenesis time courses are included. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 September 16. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript necessary to mount a proper host homeostatic response. This feedback loop is thought to be responsible for the progressive nature of type II diabetes (1).Cro...

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ~200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

Cloning of whole genomes of the genus Mycoplasma in yeast has been an essential step for the creation of the first synthetic cell. The genome of the synthetic cell is based on Mycoplasma mycoides, which deviates from the universal genetic code by encoding tryptophan rather than the UGA stop codon. The feature was thought to be important because bacterial genes might be toxic to the host yeast cell if driven by a cryptic promoter active in yeast. As we move to expand the range of bacterial genomes cloned in yeast, we extended this technology to bacteria that use the universal genetic code. Here we report cloning of the Acholeplasma laidlawii PG-8A genome, which uses the universal genetic code. We discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast. This gene was inactivated in order to clone and stably maintain the A. laidlawii genome as a centromeric plasmid in the yeast cell.

BackgroundSynthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G + C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. The model diatom Phaeodactylum tricornutum has an average G + C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast.ResultsWe present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency.ConclusionsEntire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G + C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.

A new data type called a posit is designed as a direct drop-in replacement for IEEE Standard 754 floating-point numbers (floats). Unlike earlier forms of universal number (unum) arithmetic, posits do not require interval arithmetic or variable size operands; like floats, they round if an answer is inexact. However, they provide compelling advantages over floats, including larger dynamic range, higher accuracy, better closure, bitwise identical results across systems, simpler hardware, and simpler exception handling. Posits never overflow to infinity or underflow to zero, and "Nota-Number" (NaN) indicates an action instead of a bit pattern. A posit processing unit takes less circuitry than an IEEE float FPU. With lower power use and smaller silicon footprint, the posit operations per second (POPS) supported by a chip can be significantly higher than the FLOPS using similar hardware resources. GPU accelerators and Deep Learning processors, in particular, can do more per watt and per dollar with posits, yet deliver superior answer quality.A comprehensive series of benchmarks compares floats and posits for decimals of accuracy produced for a set precision. Low precision posits provide a better solution than "approximate computing" methods that try to tolerate decreased answer quality. High precision posits provide more correct decimals than floats of the same size; in some cases, a 32-bit posit may safely replace a 64-bit float. In other words, posits beat floats at their own game.Keywords: computer arithmetic, energy-efficient computing, floating point, posits, LINPACK, linear algebra, neural networks, unum computing, valid arithmetic. Background: Type I and Type II UnumsThe unum (universal number) arithmetic framework has several forms. The original "Type I" unum is a superset of IEEE 754 Standard floating-point format [2,7]; it uses a "ubit" at the end of the fraction to indicate whether a real number is an exact float or lies in the open interval between adjacent floats. While the sign, exponent, and fraction bit fields take their definition from IEEE 754, the exponent and fraction field lengths vary automatically, from a single bit up to some maximum set by the user. Type I unums provide a compact way to express interval arithmetic, but their variable length demands extra management. They can duplicate IEEE float behavior, via an explicit rounding function.The "Type II" unum [4] abandons compatibility with IEEE floats, permitting a clean, mathematical design based on the projective reals. The key observation is that signed (two's complement) integers map elegantly to the projective reals, with the same wraparound of positive numbers to negative numbers, and the same ordering. To quote William Kahan [5]:"They typically save storage space because what you're manipulating are not the numbers, but pointers to the values. And so, it's possible to run this arithmetic very, very fast."The structure for 5-bit Type II unums is shown in fig. 1. With n bits per unum, the "u-lattice" populates the upper right quadrant of t...

Familial amyloidosis of Finnish type (FAF), or gelsolin amyloidosis, is a systemic amyloid disease caused by a mutation (D187N/Y) in domain 2 of human plasma gelsolin, resulting in domain 2 misfolding within the secretory pathway. Upon passage through the Golgi, furin endoproteolysis within domain 2 occurs as a consequence of the abnormal conformations that enable furin to bind and cleave, resulting in the secretion of a 68-kDa C-terminal plasma gelsolin fragment (amino acids173-755, C68). The C68 fragment is cleaved upon secretion from the cell by membrane type 1 matrix metalloprotease (MT1-MMP), affording the 8 and 5 kDa fragments (amino acids 173-242 and 173-225, respectively) comprising the amyloid fibrils in FAF patients. Herein, we show that the 8 and 5 kDa gelsolin fragments form amyloid fibrils by a nucleated polymerization mechanism. In addition to demonstrating the expected concentration dependence of a nucleated polymerization reaction, the addition of preformed amyloid fibrils, or "seeds", was shown to bypass the requirement for the formation of a high energy nucleus, accelerating 8 and 5 kDa D187N gelsolin amyloidogenesis. The C68 fragment can form small oligomers, but not amyloid fibrils, even when seeded with preformed 8 kDa fragment plasma gelsolin fibrils. Because the 68 kDa fragment of gelsolin does not form amyloid fibrils in vitro or in a recently published transgenic mouse model of FAF, we propose that administration of an MT1-MMP inhibitor could be an effective strategy for the treatment of FAF. † This research was supported by NIH Grant AG018917, The Skaggs Institute for Chemical Biology, and the Lita Annenberg Hazen (4 µM) that was mixed by overhead rotation (24 rpm) at 37 °C overnight. The AFM is compared to a negative control of buffer alone and a positive control of a solution of 8 kDa (4 µM) that was mixed by overhead rotation (24 rpm) at 37 °C overnight. (7) Concentration dependence of the 5 kDa plasma gelsolin fragment amyloidogenesis reaction analyzed at pH 6.5 using the TfT plate reader assay. Concentrations of 5 kDa fragment ranged from 2 µM to 40 µM, as indicated. (8) SDS-PAGE of proteinase K digested 8 and 5 kDa fibrils. This material is available free of charge via the Internet at

BackgroundPhotosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.ResultsWe adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.ConclusionsOur results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented. directed evolution | protein engineering