A conformational study of peptides with the general structure Ac-L-Xaa-Pro-D-Xaa-L-Xaa-NH_2: spectroscopic evidence for a peptide with significant β-turn character in water and in dimethyl sulfoxide

Several tetrapeptides, Ac-Val-Pro-D-Ser-His-NH_2, in particular, show significant type II β-turn character in water and in dimethyl sulfoxide. Evidence for this turn population is provided by 2D-rotating frame nuclear Overhauser effect (ROESY) spectroscopy, ^1H NMR amide temperature coefficients, and circular dichroism (CD) studies. To further investigate which residues specifically contribute to the integrity of the turn, studies on 10 tetrapeptides, having the general sequence AC-LXaa-Pro-D-Xaa-L-Xaa-NH_2, are described. The results show the effects of sequence variations on the type II β-turn forming propensity of these peptides in solution. Conclusions from these studies indicate that a cooperative effect between a sterically hindered, β-branched amino acid at the (i) position and a small, non-β-branched D-amino acid at the (i+2) position promotes turn formation. Implications for use of these sequences as structural nucleation elements in de novo protein design are discussed

Chemistry and biology of asparagine-linked glycosylation

The biosynthesis of glycoprotein conjugates is a complex process that involves the collective action of numerous enzymes. Recent research on the chemistry and biology of asparagine-linked glycosylation in our group has been focused on two specific areas. These are the development of potent inhibitors of oligosaccharyl transferase and the investigation of the conformational consequences of the glycosylation process. Since asparagine-linked glycosylation is an essential eukaryotic process, an understanding of the details of this complex transformation is of utmost importance both to fundamental biochemistry and to a consideration of the mechanisms of homeostatic control

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study.

Pan-immunoglobulin assays can simultaneously detect IgG, IgM and IgA directed against the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 S1-RBD Ig). In this work, we aim to evaluate a quantitative SARS-CoV-2 S1-RBD Ig electrochemiluminescence immunoassay (ECLIA) regarding analytical, diagnostic, operational and clinical characteristics. Our work takes the form of a population-based study in the principality of Liechtenstein, including 125 cases with clinically well-described and laboratory confirmed SARS-CoV-2 infection and 1159 individuals without evidence of coronavirus disease 2019 (COVID-19). SARS-CoV-2 cases were tested for antibodies in sera taken with a median of 48 days (interquartile range, IQR, 43-52) and 139 days (IQR, 129-144) after symptom onset. Sera were also tested with other assays targeting antibodies against non-RBD-S1 and -S1/S2 epitopes. Sensitivity was 97.6% (95% confidence interval, CI, 93.2-99.1), whereas specificity was 99.8% (95% CI, 99.4-99.9). Antibody levels linearly decreased from hospitalized patients to symptomatic outpatients and SARS-CoV-2 infection without symptoms (p < 0.001). Among cases with SARS-CoV-2 infection, smokers had lower antibody levels than non-smokers (p = 0.04), and patients with fever had higher antibody levels than patients without fever (p = 0.001). Pan-SARS-CoV-2 S1-RBD Ig in SARS-CoV-2 infection cases significantly increased from first to second follow-up (p < 0.001). A substantial proportion of individuals without evidence of past SARS-CoV-2 infection displayed non-S1-RBD antibody reactivities (248/1159, i.e., 21.4%, 95% CI, 19.1-23.4). In conclusion, a quantitative SARS-CoV-2 S1-RBD Ig assay offers favorable and sustained assay characteristics allowing the determination of quantitative associations between clinical characteristics (e.g., disease severity, smoking or fever) and antibody levels. The assay could also help to identify individuals with antibodies of non-S1-RBD specificity with potential clinical cross-reactivity to SARS-CoV-2

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50)

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50)

A Systematic Survey of Mini-Proteins in Bacteria and Archaea

BACKGROUND: Mini-proteins, defined as polypeptides containing no more than 100 amino acids, are ubiquitous in prokaryotes and eukaryotes. They play significant roles in various biological processes, and their regulatory functions gradually attract the attentions of scientists. However, the functions of the majority of mini-proteins are still largely unknown due to the constraints of experimental methods and bioinformatic analysis. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we extracted a total of 180,879 mini-proteins from the annotations of 532 sequenced genomes, including 491 strains of Bacteria and 41 strains of Archaea. The average proportion of mini-proteins among all genomic proteins is approximately 10.99%, but different strains exhibit remarkable fluctuations. These mini-proteins display two notable characteristics. First, the majority are species-specific proteins with an average proportion of 58.79% among six representative phyla. Second, an even larger proportion (70.03% among all strains) is hypothetical proteins. However, a fraction of highly conserved hypothetical proteins potentially play crucial roles in organisms. Among mini-proteins with known functions, it seems that regulatory and metabolic proteins are more abundant than essential structural proteins. Furthermore, domains in mini-proteins seem to have greater distributions in Bacteria than Eukarya. Analysis of the evolutionary progression of these domains reveals that they have diverged to new patterns from a single ancestor. CONCLUSIONS/SIGNIFICANCE: Mini-proteins are ubiquitous in bacterial and archaeal species and play significant roles in various functions. The number of mini-proteins in each genome displays remarkable fluctuation, likely resulting from the differential selective pressures that reflect the respective life-styles of the organisms. The answers to many questions surrounding mini-proteins remain elusive and need to be resolved experimentally

Problematic Stabilizing Films in Petroleum Emulsions: Shear Rheological Response of Viscoelastic Asphaltene Films and the Effect on Drop Coalescence

Adsorption of asphaltenes at the water-oil interface contributes to the stability of petroleum emulsions by forming a networked film that can hinder drop-drop coalescence. The interfacial microstructure can either be liquid-like or solid-like, depending on (i) initial bulk concentration of asphaltenes, (ii) interfacial aging time, and (iii) solvent aromaticity. Two techniques--interfacial shear rheology and integrated thin film drainage apparatus--provided equivalent interface aging conditions, enabling direct correlation of the interfacial rheology and droplet stability. The shear rheological properties of the asphaltene film were found to be critical to the stability of contacting drops. With a viscous dominant interfacial microstructure, the coalescence time for two drops in intimate contact was rapid, on the order of seconds. However, as the elastic contribution develops and the film microstructure begins to be dominated by elasticity, the two drops in contact do not coalescence. Such step-change transition in coalescence is thought to be related to the high shear yield stress (~10(4) Pa), which is a function of the film shear yield point and the film thickness (as measured by quartz crystal microbalance), and the increased elastic stiffness of the film that prevents mobility and rupture of the asphaltene film, which when in a solid-like state provides an energy barrier against drop coalescence