Protein scientists are paving the way to a new phase in protein design and engineering. Approaches and methods are being developed that could allow the design of proteins beyond the confines of natural protein structures. This possibility of designing entirely new proteins opens new questions: What do we build? How do we build into protein-structure space where there are few, if any, natural structures to guide us? To what uses can the resulting proteins be put? And, what, if anything, does this pursuit tell us about how natural proteins fold, function and evolve? We describe the origins of this emerging area of fully de novo protein design, how it could be developed, where it might lead, and what challenges lie ahead.
The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.
Molecular
recognition underpins all specific protein–ligand
interactions and is essential for biomolecular functions. The prediction
of canonical binding poses and distinguishing binders from nonbinders
are much sought after goals. Here, we apply the generalized replica
exchange with solute tempering method, gREST, combined with a flat-bottom
potential to evaluate binder and nonbinder interactions with a T4
lysozyme Leu99Ala mutant. The buried hydrophobic cavity and possibility
of coupled conformational changes in this protein make binding predictions
difficult. The present gREST simulations, enabling enhanced flexibilities
of the ligand and protein residues near the binding site, sample bindings
in multiple poses, and correct portrayal of X-ray structures. The
free-energy profiles of binders (benzene, ethylbenzene, and n-hexylbenzene) are distinct from those of nonbinders (phenol
and benzaldehyde). Bindings of the two larger molecules seem to be
associated with a structural change toward an excited conformation
of the protein, which agrees with experimental findings. The protocol
is generally applicable to various proteins having buried cavities
with limited access for ligands with different shapes, sizes, and
chemical properties.
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