Here
we show that a solvent-exposed f-position
(i.e., residue 14) within a well-characterized trimeric helix bundle
can facilitate a stabilizing long-range synergistic interaction involving b-position Glu10 (i.e., i – 4 relative
to residue 14) and c-position Lys18 (i.e., i + 4), depending the identity of residue 14. The extent
of stabilization associated with the Glu10-Lys18 pair depends primarily
on the presence of a side-chain hydrogen-bond donor at residue 14;
the nonpolar or hydrophobic character of residue 14 plays a smaller
but still significant role. Crystal structures and molecular dynamics
simulations indicate that Glu10 and Lys18 do not interact directly
with each other but suggest the possibility that the proximity of
residue 14 with Lys18 allows Glu10 to interact favorably with nearby
Lys7. Subsequent thermodynamic experiments confirm the important role
of Lys7 in the large synergistic stabilization associated with the
Glu10-Lys18 pair. Our results highlight the exquisite complexity and
surprising long-range synergistic interactions among b-, c-, and f-position residues
within helix bundles, suggesting new possibilities for engineering
hyperstable helix bundles and emphasizing the need to consider carefully
the impact of substitutions at these positions for application-specific
purposes.
Hydrocarbon stapling and PEGylation are distinct strategies for enhancing the conformational stability and/or pharmacokinetic properties of peptide and protein drugs. Here we combine these approaches by incorporating asparagine-linked O-allyl PEG oligomers at two positions within the β-sheet protein WW, followed by stapling of the PEGs via olefin metathesis. The impact of stapling two sites that are close in primary sequence is small relative to the impact of PEGylation alone and depends strongly on PEG length. In contrast, stapling of two PEGs that are far apart in primary sequence but close in tertiary structure provides substantially more stabilization, derived mostly from an entropic effect. Comparison of PEGylation + stapling vs. alkylation + stapling at the same positions in WW reveals that both approaches provide similar overall levels of conformational stability.
Many proteins have one or more surface-exposed patches
of nonpolar
residues; our observations here suggest that PEGylation near such
locations might be a useful strategy for increasing protein conformational
stability. Specifically, we show that conjugating a PEG-azide to a
propargyloxyphenylalanine via the copper(I)-catalyzed azide–alkyne
cycloaddition can increase the conformational stability of the WW
domain due to a favorable synergistic effect that depends on the hydrophobicity
of a nearby patch of nonpolar surface residues.
Coiled coils are among the most abundant
tertiary and quaternary
structures found in proteins. A growing body of evidence suggests
that long-range synergistic interactions among solvent-exposed residues
can contribute substantially to coiled-coil conformational stability,
but our understanding of the key sequence and structural prerequisites
of this effect is still developing. Here, we show that the strength
of synergistic interaction involving a b-position
Glu (i), an f-position Tyr (i + 4), and a c-position Lys (i + 8) depends on the identity of the f-position
residue, the length and stability of the coiled coil, and its oligomerization
stoichiometry/surface accessibility. Combined with previous observations,
these results map out predictable sequence- and structure-based criteria
for enhancing coiled-coil stability by up to −0.58 kcal/mol
per monomer (or −2.32 kcal/mol per coiled-coil tetramer). Our
observations expand the available tools for enhancing coiled coil
stability by sequence variation at solvent-exposed b-, c-, and f-positions and suggest
the need to exercise care in the choice of substitutions at these
positions for application-specific purposes.
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