Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α -Helical Stability and Folding Kinetics

Abstract

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

In silico-designed lignin peroxidase from Phanerochaete chrysosporium shows enhanced acid stability for depolymerization of lignin

“…The introduced salt bridges between Glu-Arg ionic pairs suggested, according to the calculated Gibbs free energy, enhanced stability of the variants under acidic pH conditions. This result agrees with a previous paper in which the thermodynamic stability between peptides containing different types of salt bridges followed the trend Glu-Arg > Asp-Lys > Glu-Lys at both neutral and acidic pH [ 37 ].…”

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“…It is known that a sequence of Asp and/or Glu residues together can destabilize the helix within the protein, because they are highly charged, and repel each other. 26 , 27 The forces of repulsion of such residues are stronger than the hydrogen bonding. Moreover, a cluster of Ile residues with their large bulky R groups tends to disrupt the α-helix structure by disrupting the hydrogen bonding within the protein.…”

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“…Of the various possibilities to build salt bridges, was shown in short peptides to be more α-helix stabilizing than [ 27 ], and both are more stabilizing than the reverse salt bridges and . salt bridges are also the most favourable for the speed of folding [ 22 ]. However, comparison of long repeats of with X being either or showed, that such peptides aggregated when two or three of the X were R [ 28 ].…”

“…SAH-domains are extremely rich in glutamate (E), lysine (K) and arginine (R) [ 4 , 6 , 13 , 14 ], which have been shown to stabilize poly-alanine peptides by charge interactions along the helix [ 15 – 20 ]. Although aspartate (D) can also form stabilizing interactions with K/R [ 21 , 22 ], aspartates occur less often than isoleucine, leucine, methionine, alanine and glutamine in predicted, highly likely SAH-domains [ 13 , 14 ]. Especially repeated patterns of four E followed by four K/R seem to stabilize α-helices, while peptides with repeats of two residues do not show helical content [ 3 , 17 , 23 ].…”