Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics  Heleen Meuzelaar, Jocelyne Vreede, Sander Woutersen  Biophysical Journal  Volume 110, Issue 11, Pages 2328-2341 (June 2016) DOI: 10.1016/j.bpj.2016.04.015 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Schematic representation of the folded conformation of six of the investigated peptides, showing the salt-bridge-forming side groups (a and b) Glu− (E) and Arg+ (R), (c and d) Asp− (D) and Arg+ (R), and (e and f) Glu− (E) and Lys+ (K). (Left column) Salt-bridging side chains are spaced four (i, i + 4) peptide units apart. Shown are peptides (i + 4)ER (top), (i + 4)DR (center), and (i + 4)EK (bottom). (Right column) Salt-bridging side chains are spaced three (i, i + 3) peptide units apart and are in reverse order. Shown are peptides (i + 3)RE (top), (i + 3)RD (center), and (i + 3)KE (bottom). Structures were optimized and rendered with Chimera (38). To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Thermal denaturation curves at 222 nm using UV CD of the different E/R peptides (a and b) (22), D/R peptides (c and d), and E/K peptides (e and f), under neutral (a, c, and e) and acidic (b, d, and f) pH conditions. The solid curves represent least-square fits to a two-state unfolding transition (48). The experimental values obtained for ΔH and Tm are listed in Table S1. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Thermal unfolding curves detected at 222 nm using UV CD of the peptides containing different types of geometrically optimized salt bridges at neutral (a) and acidic (b) pH: peptides (i + 4)ER, (i + 4)DR, and (i + 4)ER. The solid curves represent least-square fits to a two-state unfolding transition (48). To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Helical hydrogen bonds in the peptide systems. (Left) Probability distribution (p) of the number of helical hydrogen bonds (hhb) for each peptide, with the means indicated by vertical dotted lines. (Right) Distance between the donor (N) and acceptor (O), dO-N, averaged over the seven simulations (excluding the first 150 ns) as a function of the residue number of the acceptor. The dashed line at 0.35 nm indicates the distance criterion for forming a hydrogen bond. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 The fraction of salt bridges shown as bar plots for each repeat, with different colors indicating in-repeat salt bridges, out-repeat salt bridges, and no salt bridge. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Probability plots as a function of the number of helical hydrogen bonds, hhb, and the number of in-repeat salt bridges, nsb. Contour lines are drawn at intervals of p = 0.01. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Temperature-dependent equilibrium FTIR spectra in the amide I′ spectral region of peptides (a) (i + 4)ER (22), (b) (i + 4)DR, and (c) (i + 4)EK at neutral pH. FTIR difference spectra were generated by subtracting the spectrum measured at 274 K from the spectrum collected at 353 K, and reflect the changes in IR absorption upon thermal unfolding of the α-helices. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Representative data set probing the T-jump relaxation dynamics of peptide (i + 4)EK in the amide I′ region after a T-jump from 293 to 301 K. (a) Three-dimensional representation of the observed relaxation kinetics corrected for solvent absorption changes. The difference in absorption (ΔA) is presented as a function of frequency and time. (b) Transient spectra for selected delay times compared to the (scaled) FTIR difference spectrum. (c) Time course of the T-jump relaxation monitored at 1630 cm−1 (α-helix) and 1658 cm−1 (random coil). The relaxation kinetics are globally fitted to a single-exponential decay function (solid curve), ΔA(t) = A0 + A1exp(−t/τ), with an observed time constant of τ = 143 ± 4 ns. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 9 Arrhenius plots of the effective folding rates (kF,eff) of the different E/R peptides (a and b) (22), D/R peptides (c and d), and E/K peptides (e and f), measured in the absence (left) and presence (right) of salt-bridge effects. The solid lines are least-square linear fits. For better comparison, in the plots of kF,eff at neutral pH (salt-bridge effects), the fits to kF,eff at acidic pH (no salt bridges) are shown as dotted lines for peptides (i + 4)ER and (i + 3)RE (b), (i + 4)DR (d), and (i + 4)EK and (i + 3)EK (f). The values for ΔH(U→F)‡ obtained from fitting of the data to the Arrhenius equation are summarized in Table 1. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 10 (a) Arrhenius plots of the effective folding rates of peptides (i + 4) ER, (i + 4) DR, and (i + 4) EK measured in the presence of salt-bridge effects at neutral pH. The fits to kF,eff at acidic pH (no salt bridges) are shown as dotted lines. (b) Arrhenius plots of the effective unfolding rates. To see this figure in color, go online. Biophysical Journal 2016 110, 2328-2341DOI: (10.1016/j.bpj.2016.04.015) Copyright © 2016 Biophysical Society Terms and Conditions