64 research outputs found
Sequence variation in ligand binding sites in proteins
BACKGROUND: The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance. RESULTS: Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys(2)His(2 )zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies. CONCLUSION: Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites
Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins
Design and characterization of structured protein linkers with differing flexibilities
Engineered fusion proteins containing two or more functional polypeptides joined by a peptide or protein linker are important for many fields of biological research. The separation distance between functional units can impact epitope access and the ability to bind with avidity; thus the availability of a variety of linkers with different lengths and degrees of rigidity would be valuable for protein design efforts. Here, we report a series of designed structured protein linkers incorporating naturally occurring protein domains and compare their properties to commonly used Gly_4Ser repeat linkers. When incorporated into the hinge region of an immunoglobulin G (IgG) molecule, flexible Gly_4Ser repeats did not result in detectable extensions of the IgG antigen-binding domains, in contrast to linkers including more rigid domains such as β2-microglobulin, Zn-ι2-glycoprotein and tetratricopeptide repeats. This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the construction of antibodies with enhanced binding properties or other fusion proteins
Liveâcell superâresolution imaging of actin using LifeActâ14 with a PAINTâbased approach
We present directâLIVEâPAINT, an easyâtoâimplement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of directâLIVEâPAINT with an actinâbinding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that directâLIVEâPAINT can be used to image actin structures below the diffractionâlimit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells
Imaging Proteins Sensitive to Direct Fusions Using Transient PeptideâPeptide Interactions
Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making themdifficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to âź200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell superresolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-ofinterest. Here, we successfully use LIVE-PAINT to image yeastmembrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.KEYWORDS: membrane protein, proteinâprotein interaction, super-resolution microscopy, live-cell imaging, LIVE-PAINT, yeas
Exploring ethical issues associated with using online surveys in educational research
Online surveys are increasingly used in educational research, yet little attention has focused on ethical issues associated with their use in educational settings. Here, we draw on the broader literature to discuss 5 key ethical issues in the context of educational survey research: dual teacher/researcher roles; informed consent; use of incentives; privacy, anonymity, and confidentiality; and data quality. We illustrate methods of addressing these issues with our experiences conducing online surveys in educational contexts. Moving beyond the procedural ethics approach commonly adopted in quantitative educational research, we recommend adopting a situated/process ethics approach to identify and respond to ethical issues that may arise during the conduct, analysis, and reporting of online survey research. The benefits of online surveying in comparison to traditional survey methods are highlighted, including the potential for online surveys to provide ethically defensible methods of conducting research that would not be feasible in offline education research settings
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SLC35A2â CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals
Pathogenic de novo variants in the Xâ linked gene SLC35A2 encoding the major Golgiâ localized UDPâ galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2â congenital disorders of glycosylation (CDG; formerly CDGâ IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin Nâ glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2â CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2â dependent UDPâ galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wildâ type to mutant alleles in fibroblasts from affected individuals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/1/humu23731_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/2/humu23731-sup-0001-Supp_Mat__2019.2.10_.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/3/humu23731.pd
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