A major limitation of biopharmaceutical proteins is their fast clearance from
circulation via kidney filtration, which strongly hampers efficacy both in
animal studies and in human therapy. We have developed conformationally
disordered polypeptide chains with expanded hydrodynamic volume comprising the
small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged
biological polymers with biophysical properties very similar to poly-ethylene
glycol (PEG), whose chemical conjugation to drugs is an established method for
plasma half-life extension. In contrast, PAS polypeptides offer fusion to a
therapeutic protein on the genetic level, permitting Escherichia
coli production of fully active proteins and obviating in
vitro coupling or modification steps. Furthermore, they are
biodegradable, thus avoiding organ accumulation, while showing stability in
serum and lacking toxicity or immunogenicity in mice. We demonstrate that
PASylation bestows typical biologics, such as interferon, growth hormone or Fab
fragments, with considerably prolonged circulation and boosts bioactivity
in vivo.
Hyperpolarization activated and cyclic nucleotide-gated (HCN) ion channels as well as cyclic nucleotide-gated (CNG) ion channels are essential for the regulation of cardiac cells, neuronal excitability, and signaling in sensory cells. Both classes are composed of four subunits. Each subunit comprises a transmembrane region, intracellular N- and C-termini, and a C-terminal cyclic nucleotide-binding domain (CNBD). Binding of cyclic nucleotides to the CNBD promotes opening of both CNG and HCN channels. In case of CNG channels, binding of cyclic nucleotides to the CNBD is sufficient to open the channel. In contrast, HCN channels open upon membrane hyperpolarization and their activity is modulated by binding of cyclic nucleotides shifting the activation potential to more positive values. Although several high-resolution structures of CNBDs from HCN and CNG channels are available, the gating mechanism for murine HCN2 channel, which leads to the opening of the channel pore, is still poorly understood. As part of a structural investigation, here, we report the complete backbone and side chain resonance assignments of the murine HCN2 CNBD with part of the C-linker.
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