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. 2004 Oct 27;126(42):13679-84.
doi: 10.1021/ja047265o.

The helical alanine controversy: an (Ala)6 insertion dramatically increases helicity

Jasper C Lin  1 , Bipasha Barua, Niels H Andersen
Affiliations

The helical alanine controversy: an (Ala)6 insertion dramatically increases helicity

Jasper C Lin et al. J Am Chem Soc. .

Abstract

Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal alpha-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson-Roig formulation, the calculated value (wAla = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host-guest techniques or in N-terminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present.

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Figures

Figure 1
Figure 1
Trp-cage structure and the extension of the N-terminal helix by the insertion of multiple alanines.
Figure 2
Figure 2
Agreement between different cage measures is illustrated for TC11b1 and TC11b7. The peak for G11 Hα2 in TC11b1 was too broad to detect in the NMR spectra taken at 300 K and above. This is the result of exchange broadening due to microsecond time constants in the folding dynamics.
Figure 3
Figure 3
Chemical shift fraction-folded measures for TC11b1 and TC11b7.
Figure 4
Figure 4
CD melts of TC11b1, -b4, and -b7 monitored at 222 nm.
Figure 5
Figure 5
Global fold stability (ΔGU) versus x for the TC11bx alanine addition series.
Figure 6
Figure 6
(A) CD spectra of TC11b1 and TC11b7 in molar ellipticity. As expected, TC11b7 exhibits a more helical signature due to added residues of the longer helix. (B) Difference spectra between TC11b1 and TC11b7, divided by the number of alanines added (6), results in a per-alanine spectrum with a classic helix signature.
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    1. Helix propensities of different residues have been rationalized in terms of side-chain entropy differences between the random coil and helical state.6a–c Helicity increases for Xaa → Ala mutations can be attributed to a reduction in ΔSU. In addition, Ala's lack of a bulky side chain allows for stabilization of H-bonding interactions with water in the helical state.

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