AFM observation of silk fibroin on mica substrates: morphologies reflecting the secondary structures
Introduction
Silk fibroin is one of the most familiar, as well as being one of useful proteins around us, and the study of its structures and properties has a long history. Various fibroin-type proteins exist in natural silks, e.g. spider's silk, and Bombyx mori silk fibroin in particular has attracted much attention. Numerous past studies have revealed that the primary structure of B. mori fibroin has a simple and regular unit; approximately 70% of the amino-acid sequence is describable as (Gly–Ala–Gly–Ala–Gly–Ser) [1]. A resent study revealed the sequence of the repeating units of (Gly–X); Residue X is Ala (64%), Ser (22%), Tyr (10%) and so on [2].
Owing to this simple structure, fibroin has been regarded as one of the best prototypes for studying the science of protein. By contrast, the higher order structures of fibroin are somewhat complicated and have been a matter of controversy. Several models have been proposed so far for the secondary structure [3], [4]. Two different phases have been identified as major secondary structures for fibroin, so-called silk I [5] and silk II [6]. The structure of silk II has been revealed to be the anti-parallel β-sheet type [7], in which the polypeptide chains are aligned and adjacent chains are connected with hydrogen bonds (>C=O⋯HN<), while the detailed structure of silk I remains somewhat unclear, even up to the present. It has been suggested that silk I contains α-helix and random coil structures [8], [9]. Metastable silk I and the random coil structure are converted into the more stable silk II by external perturbations such as elongation and heating [10]. In addition, a new crystalline phase of fibroin, termed silk III [11], was recently discovered for B. mori fibroin. That is, a trigonal structure (threefold helical chain conformation) was identified for fibroin at an air–water interface. The microstructure and morphology of the silk fibers have been also investigated in terms of the effect of deguming etc. [12]. Thus, the structures and morphology of fibroin are subjects that are currently being actively researched [13].
These results have mainly been obtained using various spectroscopic techniques (NMR [14], etc.), X-ray diffraction measurements [15], and electron microscopy [16], [17]. To obtain further insight, another tool that enables us to directly access the microstructure and morphology of fibroin seems necessary. The atomic force microscope (AFM) is a powerful tool, since AFM gives us a wealth of direct information about morphology with nanometer spatial resolution based on a novel principle that differs significantly from conventional microscopic and spectroscopic techniques. The observation of silk threads using an AFM has already been carried out by several groups [9], [18] including the nanofibril morphology and the effect of stretching on the morphology of the threads. On the other hand, recent advances in AFM methodology have also allowed us to observe a single molecule. In the field of polymer science, individual chains on solid substrates are observable for artificial polymers and biopolymers such as proteins and DNA [19]. In most cases, mica has been chosen as a substrate due to its flatness at the atomic level [20], [21]. However, the use of such an approach for the direct observation of peptide chains in fibroin has hitherto been limited. It is noteworthy that the nanomechanics of B. mori silk fibroin has been investigated by means of single molecular force microscopy, in which a cantilever is fabricated to be connected to a single fibroin molecule [18].
In order to gain spatial resolution in the AFM, it is desirable to prepare and employ a sample in which molecular chains are developed as quasi-2-dimensional films on a solid (mica) substrate. In the present study, we succeeded in the preparation of such films on mica substrates by a simple cast method. For the quasi-2-dimensional films, we observed a network-type morphology consisting of a random coil. Other characteristic morphologies reflecting the secondary structures were found by modifying the manner of sample preparation. Also, an island-like morphology corresponding to the β-sheet structure was observed.
Section snippets
Experimental details
Commercially supplied B. mori silk fibroin (powder form, Wako) was used in the as-received state. Aqueous solutions of fibroin were prepared by the following method. Mixtures of the fibroin powder and saturated aqueous solutions of calcium chloride or lithium bromide were refluxed for several hours to dissolve the fibroin. The solutions were filtered to remove residues, and dialyzed with cellulose tubes (Wako, dialysis membrane s-8) in pure water to remove the salts. The fibroin concentration
Thick films
FTIR spectra of thick fibroin films (thickness ∼10 μm) on ZnSe substrates are shown in Fig. 1 (film A–C). The absorption bands of amide I (CO stretching) approximately 1650 cm−1, amide II (NH deformation and CN stretching) approximately 1530 cm−1, and amide III (CN stretching and NH deformation) approximately 1250 cm−1 are observable. It is well established that the peak positions of these amide bands reflect the secondary structure of fibroin [24]. The thick film (film A) has spectral
Concluding remarks
In the present study, we succeeded in the formation of ultra thin films of B. mori fibroin on mica substrates by simple cast methods. The microscopic morphologies of these specimens were revealed by means of AFM, and discussed in terms of the secondary structure of fibroin. The cast of a fibroin solution prepared with calcium chloride yielded a thin film with a network-like morphology (film D). The network is made of random coiled fibroin connected by Ca2+ ions, since the network morphology was
Acknowledgements
The authors gratefully acknowledge Prof. M. Shibayama and Dr T. Norisue for useful discussions. We also acknowledge Dr Sumida for the electrophoresis measurements. This work was partly supported by a Grant-in Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 13750669).
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