13C chemical shifts of
Recently, silk fibroin has been widely attended for its biomedical material applications, because of its excellent properties such as strength, flexibility, biocompatibility and permeability (Altman et al., 2003). Compared with the silk fibroin from silkworm, spider fibroin has higher strength and stronger toughness, but its resource is limited. So people have tried to synthesize the spider fibroin using genetic recombination method to obtain the spider fibers (Fukushima, 1998; Heslot, 1998; Zhou et al., 2001). Shao et al. (Shao & Vollrath, 2002) demonstrated that although the amino acid sequence of
The methods used to investigate the silk fibroin mainly include X-ray diffraction (XRD) (Lv et al., 2005; March et al., 1955; Qiao et al., 2009; Takahashi et al., 1999; Saitoh et al., 2004; Sinsawat et al., 2002), electron diffraction (ED) (He et al., 1999), infrared (IR) (Chen et al., 2001; Mo et al., 2006), nuclear magnetic resonance (NMR) (Yao et al., 2004; Zhao et al., 2001) and so on. Among them, nuclear magnetic resonance spectroscopy is very effective to characterize the molecular chain structures of the biopolymers (Du et al., 2003; Li et al., 2008). Asakura et al. analyzed the crystal structures of 40 proteins and their NMR chemical shifts, and established the correlation of Ala, Ser, and Gly residues between 13C chemical shifts of Cα, Cβ atoms and the dihedral angels of and of the peptide chains, which are very useful to determine the protein structure by NMR method (Asakura et al., 1999). The authors also carried out a series of solid-state 13C NMR experiments to study the peptides (AG)n, a model compound of the silk fibroin, using spin diffusion NMR and rotational echo double resonance (REDOR) techniques (Asakura, 2001, 2002 a, 2002b, 2002c, 2005a, 2005c, 2007). They found two types of the secondary structures of β-turn and β-sheet. The authors suggested that the peptides GAAS in the heavy chain of silk fibroin are one of the factors influencing the structural transition of the silk fibroin from random coil to β-sheet (Asakura et al., 2002) in which the –OH ligands of Ser residues participate in the formation of hydrogen bonds (Askura, 2002, 2005; Sato et al., 2008), and Tyr, replacing the Ser in the basic (AGSGAG)n sequence, can induce the partially disordered structure (Asakura et al., 2005). Ha et al. synthesized the non-crystalline silk peptide which contains 31 amino acid residues (GTGSSGFGPYVANGGYSGYEYAWSSESDFGT), and used high resolution 2D-NMR techniques, such as COSY, TOCSY, NOESY, ROESY and HMQC and HMBC, to study the structure of the peptide in solution. They proved that the structure of the peptide is a loop (Ha et al., 2005).
Our group has investigated the environmental influences on the conformation of silk fibroin by solid-state 13C NMR during the past few years. Moreover, we also used EPR method to study the interaction between the metal ions of Cu2+, Fe3+, Mn2+ and silk fibroin, and tried to understand the role of the metal ions in the conformation transition. We are going to review our research results in this paper.
2. Component of silk fibroin and spinning process of the
The heavy-chain fibroin is predominant component (up to 85% w/w) in
During the natural silk spinning from the spinneret of a silkworm, the conformation of the silk fibroin is converted from a soluble helical form to an insoluble β-sheet form (Magoshi et al., 1996). Magoshi et al. (Kobayashi et al., 2001; Magoshi et al., 1996) found that pH changes gradually in the lumen of the silkworm from neutral (pH 6.9) in the posterior division to weakly acidic (pH 4.8) in the anterior division adjacent to the spinneret. Meantime, the concentration of the inorganic ions such as Ca2+, K+ and Mg2+ vary within each division of the silk gland (Hossain et al., 2003; Magoshi et al., 1996), which promotes the transition from gel to sol state and then formation of the silk fiber (Kobayashi et al., 2001).
Li et al. (Li et al., 2001) used circular dichroism (CD) spectroscopy to study the conformation transition of the silk fibroin from random coil to β-sheet and the β-sheet aggregation growth. The authors suggested a nucleation-dependent aggregation mechanism for the silk spinning process as in Fig. 1. There are two steps involved in this mechanism: (a) nucleation, a rate-limited step involving the conversion of the soluble random coil to insoluble β-sheet and subsequently a series of thermodynamically unfavorable association of the β-sheet unit,
Moreover, the shearing strength plays a key role in the formation of fiber, and the cooperation with pH and metal ions is necessary in the spinning process. The natural evolution of the silkworm develops the special spinning process, leading to the excellent performance of the silk fiber. Therefore, understanding the silkworm spinning process is helpful for ones to manufacture the high performance artificial fibers.
3. Secondary structure of silk fibroin
There are mainly two typical conformations, Silk I and Silk II, in the crystalline/semi-crystalline domains of the heavy-chain silk fibroin. Kratky et al. (Kratky et al., 1950) found an unstable crystal domain approaching the spinneret, named “Silk I” which is dominated by the helix conformation, as well as another stable crystal domain in the spun fibers, named “Silk II” which is dominated by the β-sheet conformation. Valuzzi et al. (Valluzzi, 1996, 1997, 1999) found a 32-helical structure of
|Cocoon||Silk II||49.7||20.2||171.7||43.9||169.4||Saito et al., 1983|
|Degummed silk||Silk II||48.6||20.2||172.2||43.1||169.6||Zhou et al., 2001|
|R. fibroin||Silk II|
|Zhou et al., 2001|
|R. fibroin (liquid)||Silk I||50.0||16.6||175.5||42.7||171.5||Asakura et al., 1984|
Despite that the
4. Environment influences on the transition of secondary structure of silk fibroin
The spinning process of the silkworm undergoes at normal temperature, normal pressure and aqueous solution with given shearing force, pH value, metallic ion contents and protein concentration.
Asakura et al. (Asakura et al., 1984) proved that the regenerated silk fibroin has the same amino acid sequence and secondary structure as the silk fibroin present in the silk gland. Therefore, we studied the solid regenerated silk fibroin which is prepared by dissolving the silk fiber in 9.3 M KBr solution and dialyzing and then drying in air in order to mimic the spinning process of water lost. The detail preparing process of the regenerated silk fibroin follows the reference report (Li et al., 2001).
4.1. Influence of pH
We used 13C CP/MAS NMR spectroscopy to study the conformation of the silk fibroin within pH range of 5.2 - 8.0 (Xie et al., 2004). Fig. 2(A) shows 13C CP/MAS NMR spectrum of the silk fibroin. The resonance peak at 5 ~ 25 ppm in Fig. 2(B) for the Cβ of Ala residue can distinguish the helix form from the β-sheet form (Liivak et al., 1998). The lineshape of the peak can be deconvoluted into four components, Silk I at 17.0 ± 0.5 ppm and Silk II at 20.0 ±0.5 ppm, as well as transition state components, Silk I-like at 15.0 ± 0.5 ppm and Silk II-like at 21.5 ± 0.5 ppm (Zhou, 2004, 2001; Zong et al., 2004).
We define the content of total Silk II conformation as the sum of Silk II and Silk II-like conformers, which is the conformation related to the -sheet form. The dependence of total Silk II content on the pH change is shown in Fig. 3. It is found that as pH decreases, total Silk II content increases. It implies that a decrease in pH favors the conformation transition from Silk I to Silk II. The reduction in negative charge by the protonation of the amino acids may promote a refolding to a more ordered state stabilized by the hydrogen bonding between chains and accompanied by an exclusion of water. The resulting orientation of the molecules and the reduction of the intermolecular distance could promote the formation of the Silk II conformation. Such a mechanism could account for the nucleation dependency of the aggregation and secondary structural transformation of the fibroin observed
4.2. Influence of Ca2+ and Cu2+
Figure 4 demonstrates that the conformation conversion of the regenerated silk fibroin is dependent on the Ca2+ concentration (Zhou et al., 2004). The low pH (5.2) and the certain amount of Ca2+ ions (10 mg/g) favor the formation of Silk II and Silk II-related intermediate.
These results, although still lacking structural analysis in detail, may help to account for the role of pH and Ca2+ ions in the natural spinning process of the silkworms.
In addition, the high concentrations of Ca2+ ions partially inhibit the formation of Silk II-related conformation probably by introducing strong electrostatic interaction between molecular chains. It implies that, the relatively higher Ca2+ ion concentrations in the posterior division and the middle part of the middle division than that in the anterior part of the middle division in the silkworm gland (Terry et al., 2004) may prevent the premature β-sheet formation. The re-reduction of the Ca2+ ion content in the anterior division of the gland could be necessary to promote the gel to the sol transition for reducing the gel strength in native fibroin solutions and to permit it to flow through the spinning duct in the latter part of the secretory pathway (Hossain et al., 2003; Magoshi et al., 1994; Ochi et al., 2002).
In addition, we studied the Cu(II) ion influence on the silk fibroin conformation (Zong et al., 2004). From Fig. 5(A), we find that a small amount of Cu(II) addition leads to an increase in the content of total Silk II conformation and the content is highest when Cu(II) concentration is 0.36 mg/g at pH of 5.2,
Fig. 5(B) shows the EPR spectra of the Cu(II)/SF complex membranes prepared with the added Cu(II) concentration of 1.8 mg/g at pH 4.0, 5.2, 6.9, and 8.0, respectively. It is evident that the spectra are remarkably sensitive to pH variation. Table 2 summarizes the extracted parameters from the deconvoluted EPR traces, such as
|pH||Component||Relevant contents (%)||Coordination modes|
The simulated results of the EPR spectra of the Cu(II)/SF complexes at pH 8.0 and 6.9 indicate that the Cu(II) coordination with silk fibroin forms predominantly a square-planar complex with coordination modes of Cu-4N (one Cu atom coordinates four nitrogen atoms) at pH of 8.0 and Cu-3N1O (one Cu atom coordinates three nitrogen atoms and one oxygen atom) at pH of 6.9. As we know, the number of deprotonated nitrogen atoms will increase at higher pH, resulting in more possibilities for Cu(II) coordination to the nitrogen atoms.
In the amorphous domain of
4.3. Influence of K+ of Na+
K+ ion influence on the silk fibroin conformation was investigated by 13C NMR and Raman Spectroscopy (Ruan et al., 2008). Fig. 7(A) shows that, as the added [K+] increases from 0 to 3.7 mg/g, the silk fibroin conformations change partially from helix to β-sheet (Ruan et al., 2008). However, further increase of [K+] from 3.7 to 12.5 mg/g induces a decrease in total Silk II content. In addition, Fig. 7(B) shows that, as [K+] increases from 0 to 3.7 mg/g, the chemical shift of the tyrosyl Cα apical peak moves from the lower field (57.5 ± 0.5 ppm) to the higher field (55.5 ± 0.5 ppm). The change in the tyrosyl Cα chemical shift is thought changing in the environment of the tyrosine within the repetitive crystalline blocks as the fibroin conformation changes from Silk I to Silk II (Asakura et al., 2002; Taddei et al., 2004). It confirms the earlier evidence (Asakura et al., 2002; Taddei et al., 2004) that the environment of the tyrosine residues in the fibroin undergoes a change in hydrophobicity during the formation of β-sheets.
The sequence of
Combining the NMR and Raman results focused on the tyrosine change (Ruan et al., 2008), we propose a process of molecular chain movement shown in Fig. 8 as [K+] is increased. In region I of Fig. 8, as the added [K+] increases from 0 to 1.2 mg/g, the tyrosine changes from a hydrophobic environment to the environment in which there is moderate hydrogen bonding. We suggest that, when the regenerated silk fibroin is in the helix (Silk I) and/or random coil conformation, the tyrosyl groups exist in a highly hydrophobic environment. The packing of the helix structure with an intra-chain H-bond is unfavorable for the phenolic-OH hydrogen-bonding interactions (Taddi et al., 2004). An increase in [K+] may induce movement of the main chains, causing tyrosine to leave its hydrophobic environment and be exposed on the surface of protein when the fibroin is in the helix-like conformation. This change in the tyrosine environment may allow the phenolic-OH oxygen to act as an acceptor of a hydrogen atom as the serine hydroxyl group to do in the silk fibroin main chain (Taddei et al., 2004) with weakly hydrogen-bonding strength, causing the silk fibroin to undergo a secondary structural transition to the Silk II conformation. As the added [K+] increases from 1.2 to 3.7 mg/g (region II), the phenolic-OH acts progressively as an acceptor of strong hydrogen bonding, giving rise to the β-sheet-related conformation. At a [K+] of 3.7 mg/g, the hydrogen bonding between tyrosine phenolic-OH oxygen and the main chain hydrogen donor in the β-sheet conformation is strongest at the point at which the total Silk II content reaches its maximum. However, a further increase in the [K+] up to 6.2 mg/g may result in some decrease in β-sheet-like conformation and the disordered intermediate re-appears. As added [K+] increases from 6.2 to 12.5 mg/g (region III), the silk fibroin conformation is thought to return to the helix and/or random coil state. This may result from the tendency of the ions to prevent β-sheet hydrogen bond formation at high fibroin concentrations as a consequence of its chaotropic effect. In region III, the tyrosine residues have returned to a hydrophobic environment.
Moreover, we investigated Na+ ion effect on the silk fibroin conformation (Ruan et al., 2008). Samples are Na+-contained regenerated silk fibroin films. 13C CP/MAS NMR demonstrates that as the added [Na+] increases, partial silk fibroin conformation transit from helix-form to β-form at certain Na+ ion concentration which is much higher than that in
4.4. Influence of ferric and ferrous ions
Fe3+/SF and Fe2+/SF samples were studied with 13C CP/MAS NMR spectroscopy to compare the different effects of ferric and ferrous ions on the conformation of silk fibroin. The effect of ferric and ferrous ions on the Silk II contents is shown in Fig. 10 (Ji et al., 2009). Within the range 0 - 75.0 μg/g of iron ions contents, Silk II contents for Fe3+/SF and Fe2+/SF are closely comparable: slowly decreasing but lying between 17% and 22%. However, when iron ions exceed 75 μg/g, Silk II content of Fe3+/SF samples increased progressively and markedly up to about 40% at 125 μg/g of [iron] (Fig. 10-a), but that of Fe2+/SF samples only increased slightly (Fig. 10-b). It indicated that a small amount of ferric ions could maintain the ratio of [helix-form]/[β-sheet form] as constant in the silk fibroin. But if more ferric ions were added, more β-sheet structures would be formed due to the interaction of ferric ions with the specific residues in fairly conserved hydrophilic spacers in the heavy chain fibroin sequence. Once the folding template was formed, the folding process would be markedly accelerated (Gillmor et al., 1997) because of the strong hydrophobic interactions between the hydrophobic spacers in the silk fibroin, leading to the aggregation of β-sheet components. The process was demonstrated nucleation dependent (Li et al., 2001).
Fe3+/SF samples with [Fe3+] of 75.0 and 125.0 μg/g were measured by EPR spectrometer. Fe3+/SF sample with [Fe3+] of 75.0 μg/g has the lowest Silk II content while that with [Fe3+] of 125.0 μg/g has the highest Silk II content (see Fig. 10), but they have a similar EPR spectrum as shown in Fig. 11-a with [Fe3+] of 75.0 μg/g. Only one signal at
Bou-Abdallah and Chasteen (Bou-Abdallah & Chasteen, 2008) assigned the EPR signal of
4.5. Influence of Mn2+
Figure 12 shows the EPR spectra of Mn(II)/SF samples which were prepared from samples with an initial pH’s of 7.5, 6.0 and 5.2 (Deng et al., 2011). All three spectra shown in Figure 12-c’s with the added Mn(II) contents of 40.0 μg/g were almost identical, indicating that the environments of the Mn(II) ions were not pH dependent. Fig. 12-d’s show typical sextet splitting along with double peaks between the adjacent peaks, very similar to that of MnCl2 in 12 M HCl aqueous solution and in methyl alcohol solution at frozen state (T = 90 K) (Allen & Nebert, 1964), where Mn2+ ion is in the Mn(H2O)62+ complex. However, Figure 12-a’s and 12-b’s with Mn(II) contents of 4.0 and 10.0 μg/g, respectively, show somewhat pH dependency; the sextet splitting becomes evident as pH decrease from 7.5 to 5.2.
13C CP/MAS NMR of silk fibroin was studied to investigate the effect of Mn(II) ions on the conformational transition of silk fibroin. Fig. 13 (Deng et al., 2011) shows the dependence of total Silk II contents (including Silk II and Silk II-like conformations) upon the added Mn(II) content and pH. Mn(II) ions over the concentration range studied show no detectable effect on the Silk II content except for a very small but significant increase by 5% in Silk II conformation at Mn(II) content of 4.0 g/g and pH from 5.2 to 7.5. The total Silk II maximum content of 32 2% was observed at the added [Mn(II)] of 4.0 μg/g and at pH of 7.5, while the content is much lower compared with the content of 54 2% obtained by adding cupric ions to the regenerated SF (Zong et al., 2004). Besides, the EDTA-treated silk fibroin had almost the same NMR spectrum as pure silk fibroin (data not shown). These observations indicate that Mn(II) had no detectable effect on the promotion of the conformation transition in SF. Those results indicate that there are two types of Mn(II) complexes present in the silk fibroin: one is six-coordinated Mn(II)/SF complex when the content of Mn(II) is small (less than 10.0 μg/g), and the other is Mn(H2O)62+ complex which predominates at higher Mn(II) concentrations. The six-coordinated complex may be formed with the Asp, Glu and His residues in the hydrophilic spacers, promoting the Silk II conformation. In contrast, the Mn(H2O)62+ complex might stabilize the first water shell thereby tending to maintain the silk fibroin Silk I conformation, therefore leading to the pH almost hardly influencing the conformation transition of the silk fibroin. Mn(II) ions, existing in the silk gland (Zhou et al., 2005), may play a role in maintaining the appropriate balance of the secondary structure components including helix-form and -form to keep the silk fibroin stable in liquid state in the secretory pathway.
Dependence of the silk fibroin secondary structure transition upon different added metal ions is summarized in Table 3. From Table 3, Ca2+, Cu2+, K+, Fe3+ ions show the evident effect on the conformation transition of the silk fibroin, while Na+, Fe2+ and Mn2+ ions show a weak effect.
|Metal Ions||Silk I to Silk II transition||references|
|Ca2+||yes||Zhou et al., 2004|
|Cu2+||Zong et al., 2004|
|K+||Ruan et al., 2008|
|Fe3+||Ji et al., 2009|
|Na+||no||Ruan et al., 2008|
|Fe2+||Ji et al., 2009|
|Mn2+||Deng et al., 2011|
The function of protein is closely dependent on the structure of protein. The components and the secondary structures of the silk fibroin evidently affect the mechanical properties of the structural protein. Magnetic resonance methods (NMR and EPR) demonstrate that the conformation of the silk fibroin can be changed from random coil and/or helix to β-sheet under certain pH value and metal ion concentration along with the change in the coordination of silk fibroin with the metal ions as well as the change in the hydrophilic and hydrophobic environments of the protein. The results are helpful for understanding the mechanism of silkworm spinning and will give the guidance for fabricating the artificially high performance biomaterials.
The projects were supported by National Natural Science Foundation of China (Nos. 10475017, 20434010, 20673022 and 29974004). We also thank many dedicated coworkers who have contributed to the researches.