Local Structure and Dynamics of Serine in the Heterogeneous Structure of the Crystalline Domain of Bombyx mori Silk Fibroin in Silk II Form Studied by 2D ¹³C–¹³C Homonuclear Correlation NMR and Relaxation Time Observation

Abstract

The crystalline fraction (Cp fraction) of silk fibroin in silk II form from the silkworm Bombyx mori is the classic example of antiparallel β-sheet and consists mainly of Ala, Ser, and Gly. In the 13C CP/MAS NMR spectrum, the Ala Cβ, Ser Cα, and Ser Cβ peaks are asymmetric, showing the heterogeneous nature of the structure, which is shown to consist of two different packing geometries, denoted domains A and B. In this work, these peaks were resolved and assigned using 2D 13C–13C homonuclear correlation NMR spectra collected with different dipolar- assisted rotational resonance (DARR) mixing times. The local structure and dynamics of serine in the Cp fraction are discussed in detail. Model peptides of the Cp fraction, [3-13C]Ser-(AGSGAG)5 with different [3-13C]Ser labeling positions, were prepared to clarify that the individual peaks of the Ser Cβ carbons come from different domains, not from different positions within a chain. The dynamics of these individual peaks were studied by measuring 13C T 1 values, which provide information on serine side chain dynamics and thus on the formation of intermolecular hydrogen bonding through the Ser OH group. Moreover, cross peaks between domains A and B were observed in the Ala Cβ DARR spectrum with a long mixing time of 400 ms, indicating a close contact between the two geometries. We conclude that the crystalline region is heterogeneous, comprising two closely associated packing geometries that form separate but small domains.

Influence factors analysis on the formation of silk I structure

“…Crystalline dimorphism in Bombyx mori silk fibroin has been an active topic of study for several decades [9,10,23,24]. There are two known crystalline forms of silk fibroin, silk I and silk II [25].…”

“…The second polymorph, silk I, is not well characterized, because it has not been possible to obtain oriented samples. The exact structure of silk I and its poly(Ala-Gly) analogue may involve a "crankshaft" chain conformation [10]. Therefore, understanding the natural of silk I structure is an essential step to understand the natural silk spinning process.…”

“…The structure of two crystalline forms in SF, silk I and silk II, has been reported based on all kinds of experimental results [10][11][12][13]. The silk II form is characterized by an anti-parallel ␤-sheet structure [11,14].…”

“…These studies indicate that the conformation of silk I chain is a repeated ␤-turn type II that is capable of forming intra-molecular hydrogen bonds [19]. At the same time, many models with limited experimental data are proposed to describe the structure of silk I, such as crankshaft model [10], etc.…”

Characterization of water in hydrated Bombyx mori silk fibroin fiber and films by 2H NMR relaxation and 13C solid state NMR

“…The 13 C labeling of Ala Cβ carbon was performed by transamination from [3- 13 C] Ser in the silkworm [11]. The 13 C labeling sites of these amino acids are shown in Supplementary data s-2.…”