Determining the secondary and tertiary structure of natural and synthetic spider silk

Throughout evolution spiders have mastered material science as they have developed fibers with an unrivaled combination of strength and elasticity. Although the spider can produce six solid silk fibers and one aqueous silk glue, the major ampullate fiber represents a feat of natural engineering to be explored and unraveled. In addition to major ampullate silk, minor ampullate silk has mechanical properties tailored for its biological function. Both of these mechanically balanced fibers are composites of two proteins, MaSp1 and MaSp2 or MiSp1 and MiSp2 respectively. Ecological differences in species have led to differences in mechanical performance of their respective major ampullate fibers by adjusting the ratio of MaSp1 to MaSp2. There is a strong correlation between the structure and function of the proteins, particularly when considering the precise composition of recognized structural amino acid motifs. Differences in the overall protein composition of the fiber highlight the functional impact of the motifs. The secondary and tertiary structure of some of the individual amino acid domains or motifs has been determined via a variety of biophysical techniques; however, there are many portions of the silk proteins that remain unknown. Solid state Nuclear Magnetic Resonance (ssNMR) has long provided a technical approach to probe the structure of spider silk. Many ssNMR pulse sequences have been utilized to delve into the structure/function relationship of the unknown aspects of spider silk. Despite a wide variety of pulse sequences to probe different chemical environments and molecular interactions, technical limitations imposed by the repetitive nature of spider silk have limited the utility of ssNMR.;Many complex two-dimensional ssNMR pulse sequences require an enrichment of the natural isotopic amino acid abundance and thus unique amino acid interactions for label incorporation. In order to isotopically label previously obscured regions of the major ampullate silk, de novo amino acid metabolism was exploited providing a more basic and comparative understanding of spider metabolic pathways. During the course of this investigation, the rate of amino acid scrambling and isotopic placement was determined using several labeling schemes. Prior to this revelation, simple ssNMR spectra were only able to skim the surface of the proline interactions. Notably, the majority of proline in major ampullate silk is found in the GPGXX motif of MaSp2. An enriched level of label incorporation has allowed a further look into the proline region. Importantly, the chemical shifts of proline are in the same region as elastin suggesting that they both exist in beta-turns.;Not only can the secondary structure of an amino acid residue impact the physical properties but the molecular and chemical environment can effect a similar change. Water has one of the largest effects on silk, imparting the unique physical and molecular property of supercontraction. Understanding the underlying structural basis for such a biologically-relevant physical change is essential to harness the mechanics of these designer fibers. Importantly, supercontraction goes beyond the classic physical bulk mobility of the fiber exemplified by major ampullate silk and also promotes an increased molecular mobility even in the absence of bulk fiber mobility as revealed by ssNMR studies of minor ampullate silk. The gained mobility in both the major and minor ampullate silk correlates to a change in the viscoelastic nature (elastic modulus and the extensibility) of both wetted fibers.;Ultimately, the mechanical and structural properties of designer synthetic mimetics are impacted by (1) the amino acid sequence of the fiber, (2) the molecular and chemical environment of the fiber, and (3) synthetic spinning conditions. Alterations of any or all of these parameters can produce a designer fiber with desired mechanical properties. Synthetic fibers have been produced with the individual major ampullate proteins. A comparison of the mechanical properties and the structure of the synthetics were explored through ssNMR. Synthetic MaSp1 fibers, although they showed many similarities to the natural Nephila clavipes major ampullate fibers, showed distinct mechanical and structural differences based on the formulation of spinning dope, specifically the solvent used. Alternatively, synthetic MaSp2 fibers and lyophilized protein had many more differences when compared to Argiope aurantia silk. Importantly, the absence of beta-sheets suggest an interaction between MaSp1 and MaSp2 in the native fiber.;The culmination of all these studies represents a technical advancement in the field of spider silk research with far reaching consequences for future research on other repetitive structural proteins.