pH-Independence of Trialanine and the Effects of Termini
Blocking in Short Peptides: A Combined Vibrational, NMR, UVCD, and
Molecular Dynamics Study
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Abstract
Several lines of evidence now well
establish that unfolded peptides in general, and alanine in specific,
have an intrinsic preference for the polyproline II (pPII) conformation.
Investigation of local order in the unfolded state is, however, complicated
by experimental limitations and the inherent dynamics of the system,
which has in some cases yielded inconsistent results from different
types of experiments. One method of studying these systems is the
use of short model peptides, and specifically short alanine peptides,
known for predominantly sampling pPII structure in aqueous solution.
Recently, He et al. (J.
Am. Chem. Soc. 2012, 134, 1571−1576) proposed that
unblocked tripeptides may not be suitable models for studying conformational
propensities in unfolded peptides due to the presence of end effect,
that is, electrostatic interactions between investigated amino acid
residues and terminal charges. To determine whether changing the protonation
states of the N- and C-termini influence the conformational manifold
of the central amino acid residue in tripeptides, we have examined
the pH-dependence of unblocked trialanine and the conformational preferences
of alanine in the alanine dipeptide. To this end, we measured and
globally analyzed amide I′ band profiles and NMR J-coupling
constants. We described conformational distributions as the superposition
of two-dimensional Gaussian distributions assignable to specific subspaces
of the Ramachandran plot. Results show that the conformational ensemble
of trialanine as a whole, and the pPII content (χ<sub>pPII</sub> = 0.84) in particular, remains practically unaffected by changing
the protonation state. We found that compared to trialanine, the alanine
dipeptide has slightly lower pPII content (χ<sub>pPII</sub> =
0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly
model peptide. In addition, a two-state thermodynamic analysis of
the conformational sensitive Δε(T) and <sup>3</sup><i>J</i>(H<sup>N</sup>H<sup>α</sup>)(T) data obtained from
electronic circular dichroism and H NMR spectra indicate that the
free energy landscape of trialanine is similar in all protonation
states. MD simulations for the investigated peptides corroborate this
notion and show further that the hydration shell around unblocked
trialanine is unaffected by the protonation/deprotonation of the C-terminal
group. In contrast, the alanine dipeptide shows a reduced water density
around the central residue as well as a less ordered hydration shell,
which decreases the pPII propensity and reduces the lifetime of sampled
conformations