The Hierarchical Structure, Dynamics and Assembly of Spider Silks

Spider silks are biological protein polymers which are spun into fibers with an incredibly diverse range of mechanical properties and functions. Spider silks have long been recognized to have mechanical properties that rival most, if not all man-made materials. However, it is not yet possible to make synthetic spider silk fibers that mimic the impressive properties of their native counterparts. This is due in part to the hierarchical nature of spider silk, which introduces a complex organization of structures at nearly every length scale from the atomic to the micron level. The research in this dissertation has been in pursuit of understanding the structure, dynamics and assembly of silk proteins into high performance fibers at the molecular and nanoscale levels. The mechanical properties of a number of spider silks were measured and are discussed and placed intheir biological context. The underlying molecular interactions that give rise to these fibers are investigated by DLS, cryo-TEM, solution and solid-state NMR. The solution NMR work combined with cryo-TEM illustrates that silk proteins are stored as protein pre-assemblies in the silk gland and are the fundamental precursors to silk fibrils. The application of denaturant was used to disrupt these protein superstructures and understand the forces and dynamics that facilitate assembly. Finally, solid-state NMR was used to structurally characterize prey wrap spider silk, the toughest of the spider silks, and illustrate its α-helical coiled-coil hierarchical structure that helps explain this silk’s high extensibility and impressive mechanical performance

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum‐Stable Membranes with Low Leakage

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5–30‐fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time‐dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.Hybrid synthetic lipids with dramatically reduced leakage properties incorporate many structural features used by Nature to generate stable membranes. Covalent attachment of cholesterol groups to membrane‐spanning tetraether lipids makes it possible to generate stable liposomes with low permeability, while retaining the possibility to support functional biomolecules and deliver liposome‐encapsulated molecules to living cells.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/1/chem201701378.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/2/chem201701378-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/3/chem201701378_am.pd

The Hierarchical Structure, Dynamics and Assembly of Spider Silks

Spider silks are biological protein polymers which are spun into fibers with an incredibly diverse range of mechanical properties and functions. Spider silks have long been recognized to have mechanical properties that rival most, if not all man-made materials. However, it is not yet possible to make synthetic spider silk fibers that mimic the impressive properties of their native counterparts. This is due in part to the hierarchical nature of spider silk, which introduces a complex organization of structures at nearly every length scale from the atomic to the micron level. The research in this dissertation has been in pursuit of understanding the structure, dynamics and assembly of silk proteins into high performance fibers at the molecular and nanoscale levels. The mechanical properties of a number of spider silks were measured and are discussed and placed intheir biological context. The underlying molecular interactions that give rise to these fibers are investigated by DLS, cryo-TEM, solution and solid-state NMR. The solution NMR work combined with cryo-TEM illustrates that silk proteins are stored as protein pre-assemblies in the silk gland and are the fundamental precursors to silk fibrils. The application of denaturant was used to disrupt these protein superstructures and understand the forces and dynamics that facilitate assembly. Finally, solid-state NMR was used to structurally characterize prey wrap spider silk, the toughest of the spider silks, and illustrate its α-helical coiled-coil hierarchical structure that helps explain this silk’s high extensibility and impressive mechanical performance

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies

Thiol-Triggered Release of Intraliposomal Content from Liposomes Made of Extremophile-Inspired Tetraether Lipids.

Liposomal drug-delivery systems have been used for delivery of drugs to targeted tissues while reducing unwanted side effects. DOXIL, for instance, is a liposomal formulation of the anticancer agent doxorubicin (DOX) that has been used to address problems associated with nonspecific toxicity of free DOX. However, while this liposomal formulation allows for a more-stable circulation of doxorubicin in the body compared to free drug, the efficacy for cancer therapy is reduced in comparison with systemic injections of free drug. A robust liposomal system that can be triggered to release DOX in cancer cells could mitigate problems associated with reduced drug efficacy. In this work, we present a serum-stable, cholesterol-integrated tetraether lipid comprising of a cleavable disulfide bond, {GcGT(S-S)PC-CH}, that is designed to respond to the reducing environment of the cell to trigger the release intraliposomal content upon cellular uptake by cancer cells. A cell viability assay revealed that DOX- loaded liposomes composed of pure GcGT(S-S)PC-CH lipids were ∼20 times more toxic than DOXIL, with an IC50 value comparable to that of free DOX. The low inherent membrane-leakage properties of GcGT(S-S)PC-CH liposomes in the presence of serum, combined with an intracellular triggered release of encapsulated cargo, represents a promising approach for developing improved drug-delivery formulations for the treatment of cancer and possibly other diseases

Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 μM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum-Stable Membranes with Low Leakage.

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells

Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes.

This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids