24 research outputs found
Perfluoroalkyl Chains Direct Novel Self-Assembly of Insulin
The self-assembly of biopharmaceutical peptides into multimeric, nanoscale objects, as well as their disassembly to monomers, is central for their mode of action. Here, we describe a bioorthogonal strategy, using a non-native recognition principle, for control of protein self-assembly based on intermolecular fluorous interactions and demonstrate it for the small protein insulin. Perfluorinated alkyl chains of varying length were attached to desB30 human insulin by acylation of the Îľ-amine of the side-chain of LysB29. The insulin analogues were formulated with Zn<sup>II</sup> and phenol to form hexamers. The self-segregation of fluorous groups directed the insulin hexamers to self-assemble. The structures of the systems were investigated by circular dichroism (CD) spectroscopy and synchrotron small-angle X-ray scattering. Also, the binding affinity to the insulin receptor was measured. Interestingly, varying the length of the perfluoroalkyl chain provided three different scenarios for self-assembly; the short chains hardly affected the native hexameric structure, the medium-length chains induced fractal-like structures with the insulin hexamer as the fundamental building block, while the longest chains lead to the formation of structures with local cylindrical geometry. This hierarchical self-assembly system, which combines Zn<sup>II</sup> mediated hexamer formation with fluorous interactions, is a promising tool to control the formation of high molecular weight complexes of insulin and potentially other proteins
Linear Multiepitope (Glyco)peptides for Type-Specific Serology of Herpes Simplex Virus (HSV) Infections
Detection
of type-specific antibodies is an important and essential part of
accurate diagnosis, even in silent carriers of herpes simplex virus
(HSV)-1 (oral) and HSV-2 (genital) infections. Serologic assays that
identify HSV-1 and HSV-2 type-specific antibodies have been commercially
available for more than a decade but often face problems related to
cross-reactivity and similar issues. Attempts to identify type-specific
peptide epitopes for use in serology for both HSV-1 and HSV-2 have
been limited. We recently demonstrated epitope mapping of envelope
glycoprotein G2 and identified a type-specific glycopeptide epitope
that broadly recognized HSV-2 infected individuals. In the present
work we have performed a comprehensive glycopeptide synthesis and
microarray epitope mapping of 14 envelope proteins from HSV-1 and
HSV-2, namely, gB, gC, gD, gE, gG, gH, and gI, using sera from HSV-1-
and HSV-2-infected individuals and control sera. Several unique type-specific
peptide epitopes with high sensitivity were identified and synthesized
as one large linear multiepitope sequence using microwave-assisted
solid-phase (glyco)Âpeptide synthesis. Microarray validation with clinically
defined HSV and Varicella Zoster (VZV) sera confirmed excellent cumulative
specificities and sensitivities
Synergy of Two Highly Specific Biomolecular Recognition Events: Aligning an AT-Hook Peptide in DNA Minor Grooves via Covalent Conjugation to 2â˛-Amino-LNA
Two highly specific
biomolecular recognition events, nucleic acid
duplex hybridization and DNA-peptide recognition in the minor groove,
were coalesced in a miniature ensemble for the first time by covalently
attaching a natural AT-hook peptide motif to nucleic acid duplexes
via a 2â˛-amino-LNA scaffold. A combination of molecular dynamics
simulations and ultraviolet thermal denaturation studies revealed
high sequence-specific affinity of the peptideâoligonucleotide
conjugates (POCs) when binding to complementary DNA strands, leveraging
the bioinformation encrypted in the minor groove of DNA duplexes.
The significant cooperative DNA duplex stabilization may pave the
way toward further development of POCs with enhanced affinity and
selectivity toward target sequences carrying peptide-binding genetic
islands
Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary <i>N</i>âAlkyl and <i>N</i>,<i>N</i>âDialkyl Chitosan Derivatives
A highly efficient method for chemical
modification of chitosan
biopolymers by reductive amination to yield <i>N</i>,<i>N</i>-dialkyl chitosan derivatives was developed. The use of
3,6-<i>O</i>-di-<i>tert</i>-butyldimethylsilylchitosan
as a precursor enabled the first 100% disubstitution of the amino
groups with long alkyl chains. The corresponding mono <i>N-</i>alkyl derivatives were also synthesized, and all the alkyl compounds
were then quaternized using an optimized procedure. These well-defined
derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length
of the alkyl chain, but the order was dependent on the bacterial strain.
Toxicity against human red blood cells and human epithelial Caco-2
cells was found to be proportional to the length of the alkyl chain.
The most active chitosan derivatives were found to be more selective
for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium
chloride and benzalkonium chloride, as well as the antimicrobial peptides
melittin and LL-37
A Scalable High-performance Topographic Flow Direction Algorithm for Hydrological Information Analysis
Hydrological information analyses based on Digital Elevation Models (DEM) provide hydrological properties derived from high-resolution topographic data represented as an elevation grid. Flow direction is one of the most computationally intensive functions in the current implementation of TauDEM, a broadly used high-performance hydrological analysis software in hydrology community. Hydrologic flow direction defines a flow field on the DEM that directs flow from each grid cell to one or more of its neighbors. This is a local computation for the majority of grid cells, but becomes a global calculation for the geomorphologically motivated procedure in TauDEM to route flow across flat regions. As the resolution of DEM becomes higher, the computational bottleneck of this function hinders the use of these DEM data in large-scale studies. This paper presents an efficient parallel flow direction algorithm that identifies spatial features (e.g., flats) and reduces the number of sequential and parallel iterations needed to compute their geomorphologically motivated flow direction. Numerical experiments show that our algorithm outperformed the existing parallel D8 algorithm in TauDEM by two orders of magnitude. The new parallel algorithm exhibited desirable scalability on Stampede and ROGER supercomputers
AUC results for the B25C-dimer.
<p><b>A:</b> SV Analysis of the B25C-dimer in the presence of 2 Zn<sup>2+</sup>/hexamer (insulin normals). In the top part of the figure, open circles represent the g(s*)/s-curve derived from a dcdt-analysis. For clarity, only every 10<sup>th</sup> data point is shown. The solid red line represents the fit to a model of a single ideal species, resulting in the parameters shown in Tabel 2. The bottom part of the figure represents the local deviations between the experimental and simulated data (residuals). Every data point is shown. The rmsd of the shown fit is 9.83Ă10<sup>â3</sup>. <b>B:</b> Representative data of a SE experiment used to determine the self-association model of B25C. In the top part of the figure, open circles represent experimental concentration distributions at apparent thermo- and hydrodynamic equilibrium for one concentration (out of five) at 15 krpm (black), 24 krpm (red) and 36 krpm (green). For clarity, only every 10<sup>th</sup> data point is shown. The solid like-colored lines represent the global fit to all measured conditions to a model of a reversible monomer-dimer model, resulting in the equilibrium coefficient mentioned in the text. The bottom part of the figure represents the local deviations between the experimental and simulated data (residuals). Every data point is shown. The molar mass parameter was fixed to its expected value and the global rmsd of the fit is 7.4Ă10<sup>â3</sup>.</p
Measurements of <i>in vitro</i> activity of the B25C-dimer compared to HI.
<p><b>A:</b> Representative insulin receptor binding curves for HI(black), B25C-NEM1 (dark gray) B25C-NEM2(gray)and the B25C dimer(light gray). <b>B:</b> Representative metabolic dose response curves for HI(black) and the B25C-dimer (dark gray). Each point on the graph represents the mean Âą SD, nâ=â4 within one assay.</p
Neoglycolipids for Prolonging the Effects of Peptides: Self-Assembling Glucagon-like Peptide 1 Analogues with Albumin Binding Properties and Potent in Vivo Efficacy
Novel
principles for optimizing the properties of peptide-based
drugs are needed in order to leverage their full pharmacological potential.
We present the design, synthesis, and evaluation of a library of neoglycolipidated
glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug
candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation
of GLP-1 balanced the lipophilicity, directed formation of soluble
oligomers, and mediated albumin binding. Moreover, neoglycolipidation
did not compromise bioactivity, as in vitro potency of neoglycolipidated
GLP-1 analogues was maintained or even improved compared to native
GLP-1. This translated into pronounced in vivo efficacy in terms of
both decreased acute food intake and improved glucose homeostasis
in mice. Thus, we propose neoglycolipidation as a novel, general method
for modulating the properties of therapeutic peptides
Data collection and refinement statistics.
a<p>
<i>R<sub>merge</sub>â=âÎŁ|I<sub>i</sub>âI|/ÎŁI where I<sub>i</sub> is an individual intensity measurement and I is the mean intensity for this reflection.</i></p>b<p>
<i>R valueâ=âcrystallographic R-factorâ=âÎŁ|F<sub>obs</sub>|â|F<sub>calc</sub>|/ÎŁ|F<sub>obs</sub>|, where Fobs and Fcalc are the observed and calculated structure factors respectively. R<sub>free</sub> value is the same as R value but calculated on 5% of the data not included in the refinement.</i></p>c<p>
<i>Root-mean-square deviations of the parameters from their ideal values.</i></p
Cartoon representation of the crystal structure of the B25C-dimer.
<p><b>A:</b> The A chain is coloured in green and the B chain is shown in blue. The additional disulphide bond is shown by stick representation (yellow). An omit map was calculated by omitting the Sulphur atom of B25C. The resulting difference electron density Fo-Fc map is coloured in orange at Ď-levelâ=â3.0. It is clear from the structure that the two monomers are linked by a disulfide bond between the two adjoining B25C. <b>B:</b> Comparison of the B25C structure (blue) with that of the porcine in-sulin (PDB code 1B2E) (grey). The CÎą trace shows that the two structures have a high resemblance with minor deviations in CÎą positions at residue B21E and B29K.</p