4,503 research outputs found
Epitope mapping using mRNA display and a unidirectional nested deletion library
In vitro selection targeting an anti-polyhistidine monoclonal antibody was performed using mRNA display with a random, unconstrained 27-mer peptide library. After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined by constructing a high-complexity, unidirectional fragment library from the final selection pool. Selection by mRNA display minimized the dominant peptide from the original selection to a 15-residue functional sequence (peptide Cmin: RHDAGDHHHHHGVRQ; K-D = 38 nM). Other peptides recovered from the fragment library selection revealed a separate consensus motif (ARRXA) C-terminal to the histidine track. Kinetics measurements made by surface plasmon resonance, using purified Fab (antigen-binding fragment) to prevent avidity effects, demonstrate that the selected peptides bind with 10- to 75-fold higher affinities than a hexahistidine peptide. The highest affinity peptides (K-D approximate to 10 nM) encode both a short histidine track and the ARRXA motif, suggesting that the motif and other flanking residues make important contacts adjacent to the core polyhistidine-binding site and can contribute > 2.5 kcal/mol of binding free energy. The fragment library construction methodology described here is applicable to the development of high-complexity protein or cDNA expression libraries for the identification of protein-protein interaction domains
Growth and Examination of Calcium Single Crystals
This report is in two sections. The first section relates to a vacuum technique for growing single crystals of calcium. The second section discusses the results of an X-ray examination of crystals of zinc by the back reflection Laue method
Dynamic Pattern Formation in a Vesicle-Generating Microfluidic Device
Spatiotemporal pattern formation occurs in a variety of nonequilibrium physical and chemical systems. Here we show that a microfluidic device designed to produce reverse micelles can generate complex, ordered patterns as it is continuously operated far from thermodynamic equilibrium. Flow in a microfluidic system is usually simple—viscous effects dominate and the low Reynolds number leads to laminar flow. Self-assembly of the vesicles into patterns depends on channel geometry and relative fluid pressures, enabling the production of motifs ranging from monodisperse droplets to helices and ribbons
A Peptide Core Motif for Binding to Heterotrimeric G Protein α Subunits
Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G{alpha}i1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G{alpha}i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G{alpha} subunits representing all four G protein classes, acting as a core motif for G{alpha} interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G{alpha}i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G{alpha}i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G{alpha} subunits and excludes association with G{beta}{gamma}. R6A-G{alpha}i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg2+, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G{alpha}i1/G{alpha}s chimeras identify two regions of G{alpha}i1 (residues 1–35 and 57–88) as determinants for strong R6A-Gi{alpha}1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states
Low self-esteem prospectively predicts depression in adolescence and young adulthood
Low self-esteem and depression are strongly correlated in cross-sectional studies, yet little is known about their prospective effects on each other. The vulnerability model hypothesizes that low self-esteem serves as a risk factor for depression, whereas the scar model hypothesizes that low self-esteem is an outcome, not a cause, of depression. To test these models, the authors used 2 large longitudinal data sets, each with 4 repeated assessments between the ages of 15 and 21 years and 18 and 21 years, respectively. Cross-lagged regression analyses indicated that low self-esteem predicted subsequent levels of depression, but depression did not predict subsequent levels of self-esteem. These findings held for both men and women and after controlling for content overlap between the self-esteem and depression scales. Thus, the results supported the vulnerability model, but not the scar model, of self-esteem and depression
The Emergence of Competition Between Model Protocells
The transition from independent molecular entities to cellular structures with
integrated behaviors was a crucial aspect of the origin of life. We show that simple
physical principles can mediate a coordinated interaction between genome and
compartment boundary, independent of any genomic functions beyond self-replication.
RNA, encapsulated in fatty acid vesicles, exerts an osmotic pressure on
the vesicle membrane that drives the uptake of additional membrane components,
leading to membrane growth at the expense of relaxed vesicles, which shrink. Thus,
more efficient RNA replication could cause faster cell growth, leading to the
emergence of Darwinian evolution at the cellular level
A General Route for Post-Translational Cyclization of mRNA Display Libraries
Cyclic peptides are attractive scaffolds for the design of conformationally constrained molecular therapeutics. Previously, biological display libraries could only be cyclized via disulfide bonds, which are labile and can be reduced in an intracellular environment. In this paper, we construct high diversity, covalently cyclized mRNA display libraries (>10^(13) sequences) and analyze the cyclization reaction using MALDI-TOF MS and unnatural amino acid incorporation. Our route allows the extent of cyclization to be evaluated quantitatively and is broadly applicable to a variety of cyclization chemistries
Spinal V2b neurons reveal a role for ipsilateral inhibition in speed control
The spinal cord contains a diverse array of interneurons that govern motor output. Traditionally, models of spinal circuits have emphasized the role of inhibition in enforcing reciprocal alternation between left and right sides or flexors and extensors. However, recent work has shown that inhibition also increases coincident with excitation during contraction. Here, using larval zebrafish, we investigate the V2b (Gata3+) class of neurons, which contribute to flexor-extensor alternation but are otherwise poorly understood. Using newly generated transgenic lines we define two stable subclasses with distinct neurotransmitter and morphological properties. These V2b subclasses synapse directly onto motor neurons with differential targeting to speed-specific circuits. In vivo, optogenetic manipulation of V2b activity modulates locomotor frequency: suppressing V2b neurons elicits faster locomotion, whereas activating V2b neurons slows locomotion. We conclude that V2b neurons serve as a brake on axial motor circuits. Together, these results indicate a role for ipsilateral inhibition in speed control
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