Quantifying the fraction of glycine and alanine in beta-sheet and helical conformations in spider dragline silk using solid-state NMR

Abstract

Solid-state two-dimensional refocused INADEQUATE MAS NMR experiments resolve distinct helical and beta-sheet conformational environments for both alanine and glycine in Nephila clavipes dragline silk fibers; the fraction of alanine and glycine in beta-sheet structures is determined to be 82% +/- 4% and 28% +/- 5%, respectively.

Fig. 1

The 2D ¹³C homonuclear through-bond DQ/SQ spectrum of native (dry) Nephila clavipes spider dragline silk obtained with the refocused INADEQUATE pulse sequence. Data were collected on an 800 MHz spectrometer with a 1 ms CP contact time and a 40 kHz MAS frequency. The spectral width was 62.5 kHz in both dimensions and 256 t₁ increments were collected in the indirect dimension with a 3 s recycle delay. The connectivity and assignment agrees with previous ¹³C correlation spectra collected with through-space dipolar recoupling.

Fig. 2

The 2D ¹³C homonuclear through-bond DQ/SQ spectrum of Nephila clavipes spider dragline silk plasticized with water. The refocused INADEQUATE pulse sequence was used with ¹³C direct excitation. Data were collected on a 400 MHz spectrometer with a 20 kHz MAS frequency. The spectral width was 50 kHz in both dimensions and 288 t₁ increments were collected in the indirect dimension with a 1 s recycle delay. The short recycle delay enhances the signal from mobile amino acids.

Fig. 3

The carbonyl region of the refocused INADEQUATE spectrum of Nephila clavipes spider dragline silk plasticized with water. The recycle delay was (a) 1 s and (b) 3 s. Mobile helical regions are enhanced in (a), while the rigid β-sheet domains are apparent in (b).