The basic repeats of the two small chimeric Flag/MaSp 2 proteins used in this study were based on the consensus repeat published for Nephila clavipes’
dragline MaSp 2 protein,4
thus combined the same number of proline-containing pentapeptides with a linker-polyalanine segment. However, instead of the MaSp 2 like (GPGXX)4
, the presumed elastic motifs under study were two smaller variants of the [GPGGX]n
found in the Flag protein (n= 4 instead of n>43) making up the highly extensible capture spiral.4
To investigate the functional role of each of these two shorter Flag-like motifs, synthetic fibers were formed in optimum, but drastically different conditions adapted to each chimeric protein. The A1S820 protein was spun into fibers using organic solvents during an automated extrusion process while the Y1S820 protein spontaneously assembled into a surface film upon elution in aqueous buffers, and this film was used to hand draw fibers. The length and thickness of these Y1S820 fibers could not be controlled as they depended on the area and local thickness of the formed film.
Both types of Flag/MaSp 2 fibers were subjected to sequential post-processing techniques that combined pre- or post-treatment with water and single fiber stretching at similar draw ratios (2–2.5x). As a result, both chimeric fiber types had greatly enhanced and more refined mechanical properties. Our data suggest that despite their clearly different modes of fiber formation and sequences of processing, both fully processed Flag/MaSp 2 protein fibers exhibited the same range of mechanical properties, thus emphasizing the prevalent role of the overall structure of their basic consensus repeat and their molecular weight in dictating fiber mechanical performance. Similar observations about the effect of polymer size on the mechanical properties were also made for two different types of synthetic MaSp 1 like fibers.30, 33
The functional role of these two Flag-like structural motif variants in the context of the MaSp 2-like basic repeat structure is best exemplified by the mechanical performances achieved by the toughest synthetic fibers produced from the two chimeric proteins after undergoing similar drawing ratios (2–2.5x), followed or preceded by fiber water treatment. These best performances for each fiber category also hinted to the optimum potential of these particular chimeric silk protein fibers under these specific spinning and processing conditions (). Despite the slight but consequential differences of Flag-like (GPGGX)4
structural motifs used in these proteins, the best fibers for both categories achieved the same extensibility (80%), while showing similar high increases in ultimate tensile strength and initial modulus. In the most controlled experimental settings for fiber generation like those described for the spun and processed A1S820
, the toughness of the fully processed fiber reached 93.5 MJ/m3
, thus was 1.6–1.7 fold lower than those of native dragline and flagelliform silks. The highest level of extensibility observed for the fully processed Flag-MaSp 2 fibers compares well to the data obtained for processed fibers spun from a recombinant ADF-3 protein encoded by a native partial MaSp 2 sequence despite the use of very different spinning and processing conditions, i.e.
the use of methanol for coagulation and fiber double drawing with methanol and water.34
Additionally, besides showing the compatibility of these Flag-like elastic motifs with the MaSp 2 like crystalline domains in the hybrid chimeric protein, the roles of the short Flag-like motifs in providing extensibility to the fiber were preserved despite the rigidity of the basic repeat, and the lesser number of elastic-like motifs present compared to that present in the native-like ADF3 sequence61
Mechanical properties of the toughest synthetic Flag/MaSp 2 fibers.
The original combination of the Flag motifs with the MaSp 2 dragline silk crystalline motif in the case of the Y1S820
protein also provided a unique self-assembly ability in aqueous solutions similar to that observed for other synthetic MaSp 1 and 2 recombinant proteins containing native C- and/or N-termini sequences.23–25
This result shows that silk protein re-design, or rather the reengineering of non β-sheet crystalline forming motifs in the synthetic consensus repeat, can also be used to control protein self-aggregation. NMR data on the lyophilized Y1S820
film showed formation of β-sheet structures in the polyalanine regions in aqueous environments while the lyophilized Y1S820
protein, which is highly hygroscopic, is primarily disordered and in a random coil geometry. Water seemed to have promoted the crystallization of the polyalanine motifs which drove the spontaneous assembly mechanism seen for this specific Y1S820
chimeric protein in aqueous conditions. Furthermore, the mechanical data between the as-pulled versus processed Y1S820
-P fibers, though highly variable, due to the less standardized mode of fiber generation, showed that the successive 2x drawing into the air of these water-saturated fibers was responsible for increases in fiber stiffness and ultimate strength, probably due to an increase in crystallite orientation though no XRD data could be collected due to the high salt content of this fiber. As a result of this treatment, the processed fibers were significantly tougher. Mechanically, the processed Y1S820
-P fibers had a similar stress/strain response as that seen for native dragline silk1–3
. For these fibers formed in aqueous environments, it is difficult to separate the role of water versus that of stretching in improving mechanical properties. However, due to the inability to process the Y1S8 as-spun fibers, these processed Y1S820
-P fibers were valuable to compare to the A1S820
chimeric protein fibers since SEM data showed that they seemed to have attained similar levels of fiber organization.
The mechanical performances of these processed Y1S820
-P fibers depended on fiber diameter, and seemed best and more reproducible for fibers with diameters ranging between 10–15 μm. This optimum fiber diameter range is 2–3 times larger than those of native silk fibers while the size of the recombinant protein is 5-fold smaller. It is important to note that chemical polymer-based fibers,62–63
and natural protein-based fibers such as spider and silkworm silks, as well as wools64–65
also have variable diameters leading to variation in their mechanical properties though these are much less pronounced than what is seen for synthetic fibers. As a general rule, tensile strength and moduli increase when the diameter of the fiber decreases. This tendency was also observed for fibers produced by extrusion of spinning dopes made of resolubilized spider or silkworm silks,31, 66
bovine Achilles tendons (collagen),67
or for fibers generated from self-assembled non fibrous protein films.68
Additionally, in combination with the effect of fiber diameter, the presence of imperfections or flaws, a still hollow core, and the solidification of trapped salts in the dried Y1S820
-P fibers, most likely contributed to the more random breakage of the fibers. While the Y1S820
control fibers had already an initial extremely high extensibility, probably due to their saturated water content, it is evident that the major increase in extensibility seen throughout the processing of the A1S820
spun fibers generated in a more controlled experimental setting, only reached its maximum in the last processing step which consisted in full water-treatment while physically constraining the pre-stretched fiber. This further confirms the similarity in behavior of both chimeric proteins and native spider silk proteins for which water is a well-known plasticizer that affects both amorphous and crystalline regions41, 52
allowing molecular rearrangements in the fiber during post-spinning treatments.
In the case of the A1S820 fibers, we provided a complete morphological (SEM) and structural analysis (NMR and XRD) from the lyophilized protein stage throughout the spinning and different processing treatments to understand the nature and origin of the molecular transformations and internal network reorganization that are associated with the observed evolving mechanical properties of the fibers. NMR data showed that the spinning process drove β-sheet formation in the polyalanine regions for the A1S820 protein since the as-spun fibers had a higher β-sheet content than that seen in the lyophilized protein, but it failed to give the same result for the Y1S820 protein. SEM data showed that the imperfect thick A1S820 as-spun fibers experienced very clean or sharp transversal breaks that are characteristic of an extremely poor fiber internal network organization. However, these A1S820 as-spun fibers were not obviously spongy or porous like the Y1S820 ones which shattered when dry.
The fact that once wetted, the Y1S820 as-spun fibers did show the presence of formed β-sheet structures reinforces the argument that this protein is more suited for fiber formation in aqueous environments. This clearly shows the unique capacity of the (GPGGA GPGGA)2 or A1 motif to better accommodate the structural transition of the polyalanine segment into β-sheet structures in this chimeric protein as a result of mechanical shearing and dehydration due, respectively, to extrusion and coagulation in organic conditions. Thus again, these findings show the impact of the primary structure of the silk-like protein on its spinnability under given fiber formation conditions.
XRD data revealed a disordered and overall amorphous structure for the A1S820
as-spun fibers. This isotropic characteristic indicates the presence of poorly stacked β-sheet structures that have no preferential orientation, thus further explained the brittleness of the fiber. In both case, the Flag-like elastic motifs, may also have been negatively affected in the drying fiber, contributing to further fiber brittleness. This is particularly likely since native Flag proteins are naturally hydrated and the fiber shows major decreases in mechanical properties when dried.59
After stretching the A1S820
as-spun fibers in 90% IPA at the maximum draw ratio possible (2–2.5x), the A1S820
fibers became insoluble in water. The stretched fibers also displayed impressive increases in ultimate strength and initial modulus. SEM data showed that morphologically, the fibers were more uniform and denser with more rugged breaking points. NMR data support that these morphological and mechanical changes are the results from an increase in β-sheet structures while XRD data (data not shown) indicated that it was concomitant with a slight alignment of the nanocrystals parallel to the fiber axis although the vast majority of the structure remained disordered. Such correlation between increased tensile strength and initial modulus as a result of crystal orientation during post spin draw is in agreement with previous findings for dragline silk40
and regenerated dragline silk fibers.31
However, although these A1S820
stretched fibers were still characterized by both higher extensibilities and toughness, these were extremely variable indicating the need for further processing to refine their core structure. The last step of the treatment consisted in wetting the fiber under tension in water. As revealed by NMR and XRD data, not only the A1S820
proteins in the water-treated fibers experienced an increase in β-sheet structures in their polyalanine regions but also displayed a more pronounced anisotropy indicating the packing and 3-dimensional growth of these β-sheet nanocrystals in a similar motif to that found in spider silk fibers.13, 57
Water essentially appears to help orientate the β-sheet crystals together, probably through hydrogen bonding, thus allowing them to find a stable ordered configuration. These results are in line with findings for regenerated spider silks.31
These structural changes translated mechanically in much tougher fibers that achieved maximal performances in ultimate strength, initial modulus with much higher and less variable extensibilities. Water acted as a lubricant of the polymer chains for the entire fiber network thus allowing drastic, though incomplete, fiber network reorganization. SEM data showed that these A1S820
fibers were clearly much denser, and had the highest level of fiber internal organization with both visible longitudinal microfibrils and bundle-type structures running parallel to the fiber axis. However, hollow cavities were still visible along the fiber axis, thus showing a very loosely organized and poorly interwoven fiber network. This observed fiber internal organization was reminiscent of that observed for the more flexible inner core structure seen in native dragline silk, which is uniquely composed of MaSp 2 proteins26
and also characterized by the numerous presence of canniculis
or longitudinal interspaces. It is possible, as suggested for the role of the flexible native MaSp 2 fiber inner core, that the large longitudinal interspaces in the synthetic fibers could have accounted for their ability to absorb and retain water. Thus, the remaining variability seen for the extensibility of these synthetic fibers could stem from slight differences in fiber network organization and composition between fibers. Indeed, from our study, we can hypothesize that crystallinity and/or crystal orientation levels could potentially affect the water absorption and retention levels by the fiber and thus, result in differences in fiber network plasticization that would come in play in their extensibility.
For these specific types of chimeric fibers, longer water-treatments, and/or additional fiber stretching after water-treatment may provide the additional structural changes needed to increase fiber density and refine fiber mechanical properties.