Self-assembly and soluble aggregate behavior of computationally designed coiled-coil peptide bundles

Abstract

Coiled-coil peptides have proven useful in a range of materials applications ranging from the formation of well-defined fibrils to responsive hydrogels. The ability to design from first principles their oligomerization and subsequent higher order assembly offers their expanded use in producing new materials. Toward these ends, homo-tetrameric, antiparallel, coiled-coil, peptide bundles have been designed computationally, synthesized via solid-phase methods, and their solution behavior characterized. Two different bundle-forming peptides were designed and examined. Within the targeted coiled coil structure, both bundles contained the same hydrophobic core residues. However, different exterior residues on the two different designs yielded sequences with different distributions of charged residues and two different expected isoelectric points of pI 4.4 and pI 10.5. Both coiled-coil bundles were extremely stable with respect to temperature (Tm > 80 C) and remained soluble in solution even at high (millimolar) peptide concentrations. The coiled-coil tetramer was confirmed to be the dominant species in solution by analytical sedimentation studies and by small-angle neutron scattering, where the scattering form factor is well represented by a cylinder model with the dimensions of the targeted coiled coil. At high concentrations (5-15 mM), evidence of interbundle structure was observed via neutron scattering. At these concentrations, the synthetic bundles form soluble aggregates, and interbundle distances can be determined via a structure factor fit to scattering data. The data support the successful design of robust coiled-coil bundles. Despite their different sequences, each sequence forms loosely associated but soluble aggregates of the bundles, suggesting similar dissociated states for each. The behavior of the dispersed bundles is similar to that observed for natural proteins.

Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes

“…Clustering of Atg40 was also induced by expression of Atg8 fused with a coiled-coil sequence that forms a tetramer (Atg8-CC tetramer ) ( Supplementary Fig. 4a) 25,26 . Remarkably, Yop1 and Rtn1, but not DsRed-HDEL, were enriched in the 4 × Atg8-and Atg40-positive structures ( Fig.…”

“…Plasmids for expression of fusion proteins for crystallization were constructed by inserting the genes encoding Atg40 237-252 , human FAM134B 450-468 , human SEC62 361-376 with the T367D mutation, and human RTN3 245-264 isoform e into upstream of the sequence encoding Atg8 K26P of pGEX6P-Atg8 (the K26P mutation was introduced for stabilizing Atg8) or upstream of the sequence encoding GABARAP of pGEX6P-GABARAP (with the F3S V4T mutations for enhancing crystallization). For phase-separation experiments, the mCherry and BNDL1 25,26 sequences were inserted into upstream and downstream of the ATG8 gene in pGEX6P-Atg8 K26P, respectively. pGEX-SNAP-40C was constructed by the removal of the HRV 3C protease recognition site and the insertion of the SNAP sequence from pSNAP-tag(T7) vector (New England Biolabs) with a linker (Gly-Gly-Gly-Ser-Gly-Gly-Gly) and Atg40 194-256 following a linker (Ser-Ala-Ser-Ser) to upstream and downstream of GST in pGEX6P-1 vector, respectively.…”

Searching for Aquamelt Behavior among Silklike Biomimetics during Fibrillation under Flow

“…As a result, the SAXS/WAXS patterns of the fibers are obtained. Previously, the SASSIE Web server has been applied to generate the all-atomic models of biomacromolecules − and to investigate the structuring in synthetic polymers, , including aquamelts of PEO . In our study, the SAXS/WAXS patterns of peptide fibers are compared with available experimental data. − …”

Flow-Induced Formation of Thin PEO Fibers in Water and Their Stability After the Strain Release

“…Such an approach provided us with small- and wide-angle X-ray scattering (SAXS/WAXS) patterns of the fibers at different relaxation times, which are very useful for further comparison with available experimental data. The SASSIE Web server is usually applied to generate the all-atomic models of biomacromolecules or biomolecular complexes and to determine solution structures that have scattering profiles consistent with experiments. − This technique of fitting scattering data has previously been found to be successful in the study of structures of synthetic polymers including bottlebrush chains, or designed bundles of biomimetics . However, to the best of our knowledge, PEO-containing systems have not been investigated using such an approach.…”