Temperature- and length-dependent energetics of formation for polyalanine helices in water: assignment of w(Ala)(n,T) and temperature-dependent CD ellipticity standards

Abstract

Length-dependent helical propensities w(Ala)(n,T) at T = 10, 25, and 60 degrees C are assigned from t/c values and NMR 13C chemical shifts for series 1 peptides TrpLys(m)Inp2(t)Leu-Ala(n)(t)LeuInp2Lys(m)NH2, n = 15, 19, and 25, m = 5, in water. Van't Hoff analysis of w(Ala)(n,T) show that alpha-helix formation is primarily enthalpy-driven. For series 2 peptides Ac-Trp Lys5Inp2(t)Leu-(beta)AspHel-Ala(n)-beta-(t)LeuInp2Lys5NH2, n = 12 and 22, which contain exceptionally helical Ala(n) cores, protection factor-derived fractional helicities FH are assigned in the range 10-30 degrees C in water and used to calibrate temperature-dependent CD ellipticities [theta](lambda,H,n,T). These are applied to CD data for series 1 peptides, 12 < or = n < or = 45, to confirm the w(Ala)(n,T) assignments at T = 25 and 60 degrees C. The [theta](lambda,H,n,T) are temperature dependent within the wavelength region, 222 +/- 12 nm, and yield a temperature correction for calculation of FH from experimental values of [theta](222,n,T,Exp).

Figure 1

Red dots correspond to t/c data for the series Ac–Hel–Ala_n–^tLInp₂–Lys₄Trp–NH₂ n = 4–14, in water at 25 °C. Lines plot t/c values L–R-modeled from recursively assigned w_Ala(n,25) values. Previous parameter values at 2 °C were used to model the upper blue line: A = 0.80; B = 0.17, the initiation parameter for the t-state. The lower blue line and the best-fit green correspond to B = 0.15 and 0.16; j = 6.5, the N-capping Hel parameter for cs-state helices.

Figure 2

Temperature-dependent helical propensities w_Ala(n,T) for alanine residues in water within polyalanine contexts 1a,b; blue: T = 2 °C; cyan: T = 10 °C; green: T = 25 °C; red: T = 60 °C. Values assigned at or below 25 °C show a positive length dependence, but values assigned at 60 °C show a negative length dependence. CD data (shown below) show that for n > 25, all w_Ala(n,T) converge to an upper limit.

Figure 3

(a) Experimental per-residue molar ellipticities [θ]_λ,22,T,Exp, units, for the peptide Ac–Hel(Ala₄Lys)₄Ala₂NH₂ in 32 mol % ethylene glycol–water at intervals of 10 °C from −20 °C (top green curve) to −50 °C (bottom blue curve). Convergence to temperature-independent values of [θ]_λ is observed, except within the λ region: 222 ± 12 nm; the slope at 222 nm is +260 deg cm² K⁻¹ dmol⁻¹. (b) Calculated values of [θ]_λ,H,n,T for the completely helical conformations of the series 2 peptide ^βAspHel–Ala₂₂–beta in water at 2 (blue curve), 10 (green curve), and 30 °C (red curve). Units: 10³ deg cm² dmol⁻¹.

Figure 4

Temperature dependences of Figure 3b per-residue molar ellipticities [θ]_222,H,22,T (blue) and [θ]_208,H,22,T (green) for series 2 Ala₂₂. Linear temperature regressions for Ala₂₂: at 222 nm: intercept −49.7, slope +260; at 208 nm: intercept −34.6, slope +110. Linear regressions for Ala₁₂ (data not shown): at 222 nm, intercept −44.5, slope +280; at 208 nm, intercept −34.7, slope +92. Units: intercept, 10³ deg cm² dmol⁻¹; slope, deg cm² dmol⁻¹ K⁻¹. Error limits are calculated from errors in FH values.

Figure 5

CD tests of w_Ala(n,T) data sets using [θ]₂₂₂ data (green, 25 °C; red, 60 °C) measured in water for Ala_n 1a peptides.6 L–R modeling of [θ]₂₂₂ at 25 °C (green line) used [θ]_222,n,25 = (−60 500 + 260 (25−2))(1 − X/n), X = 6.5, as assigned at 2 °C. (fit: n = 18–45, SD: 1.2). At 60 °C (red line), [θ]_222,n,60 = (−60 500 + 260 (60−2))(1 − X/n), X = 2.5. (fit: n = 16–45, SD: 0.5). Alternative (not shown): [θ]_222,n,60 = (−60 500 + 260 (25−2))(1 − X/n), X = 3.7. (n = 16–45, SD: 0.9). Units: 10³ deg cm² dmol⁻¹.

Figure 6

(a) A dual-wavelength parametric plot of cap-corrected [θ]₂₂₂ vs [θ]₂₀₈ measured in water for the series 1a peptide. Temperature is the implicit variable, and its range is 60–2 °C (respectively low and high −[θ] values), at intervals of 5 °C from 60 to 5 °C. Three local slopes are shown; 1.5 red; 1.8 green; 2.5, blue; each defined by three or four successive data points at high, medium, and low temperatures. The continuously increasing local slopes reflect the heterogeneity of helical lengths within the conformational ensemble. (The Ala₂₈ spectra exhibit a 203 nm isoelliptic point.6) (b) Series 2 peptide ^βAspHel–Ala₂₂–beta plot exhibits two regions of constant slope: 60–35 °C (red line and data points; C. C. = 0.997; slope 1.8) and 30–2 °C (blue line and data points; C. C. = 0.996; slope 3.2). Units: 10³ deg cm² dmol⁻¹.

Figure 7

Parameter dependences of Lifson–Roig-modeled fractional helicities FH for Ala₂₂ and Ala₁₂ peptides. The temperature dependence of FH values is modeled by varying the length-independent values for w. Magenta lines are calculated for Ala₂₂, orange lines for Ala₁₂. Continuous lines correspond to FH for strong cap stabilization: j = 200, c = 7; broken lines correspond to weak cap destabilization: j = c = 0.5. Symbols drawn as squares (Ala₂₂) and circles (Ala₁₂) report FH calculated from the w_Ala(n,T) of Figure 2: red: T = 60 °C, green: T = 25 °C, blue: T = 2 °C. Filled symbols correspond to j = 200, c = 7; open symbols correspond to j = c = 0.5. Symbols have been positioned on appropriate line sites at which FH values are equal; thus, an average w (and temperature) is assigned to the w_Ala(n,T)-derived FH. These sites thus define approximate fixed-w assignments for the four peptides.