Intrinsically disordered proteins access a range of hysteretic phase separation behaviors.

The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors. Experimentally and through simulations, we show that hysteresis in IDPPs is tunable and that it emerges through increasingly stable interchain interactions in the insoluble phase. To explore the utility of hysteretic IDPPs, we engineer self-assembling nanostructures with tunable stability. These findings shine light on the rich phase separation behavior of IDPs and illustrate hysteresis as a design parameter to program nonequilibrium phase behavior in self-assembling materials

Switchable Elastin-Like Polypeptides that Respond to Chemical Inducers of Dimerization

Elastin-like polypeptides (ELPs) are protein polymers that reversibly phase separate in response to increased temperature, pressure, concentration, ionic strength, and molecular weight. If it were possible to engineer their phase separation to respond to specific molecular substrates, ELP fusion proteins might be engineered as biosensors, smart biomaterials, diagnostic imaging agents, and targeted therapies. What has been lacking is a strategy to design ELPs to respond to specific substrates. To address this deficiency, we report that ELP fusion proteins phase separate in response to chemical inducers of dimerization (CID). The rationale is that ELP phase separation depends on molecular weight, concentration, and local hydrophobicity; therefore, processes that affect these properties, including noncovalent dimerization, can be tuned to produce isothermal phase separation. To test this hypothesis, constructs were evaluated consisting of an immunophilin: human FK-506 binding protein 12 (FKBP) attached to an ELP. Under stoichiometric binding of a CID, the fusion protein homodimerizes and triggers phase separation. This dimerization is reversible upon saturation with excess CID or competitive binding of a small lipophilic macrolide to FKBP. By modulating the ELP molecular weight, phase separation was tuned for isothermal response to CID at physiological ionic strength and temperature (37 °C). To interpret the relationship between transition temperature and equilibrium binding constants, an empirical mathematical model was employed. To the best of our knowledge, this report is the first demonstration of reversible ELP switching in response to controlled dimerization. Due to its simplicity, this strategy may be useful to design ELP fusion proteins that respond to specific dimeric biological entities

Inducible Fibril Formation of Silk-Elastin Diblocks

Silk-elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk-elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk-elastin diblock polypeptides on fibril formation. For silk-elastin diblocks ESm-SQn, in which the elastin pentamer repeat is ES = GSGVP and the crystallizable silk octamer repeat is SQ = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks (m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation

Micellar Self-Assembly of Recombinant Resilin-/Elastin-Like Block Copolypeptides

Reported here is the synthesis of perfectly sequence defined, monodisperse diblock copolypeptides of hydrophilic elastin-like and hydrophobic resilin-like polypeptide blocks and characterization of their self-assembly as a function of structural parameters by light scattering, cryo-TEM, and small-angle neutron scattering. A subset of these diblock copolypeptides exhibit lower critical solution temperature and upper critical solution temperature phase behavior and self-assemble into spherical or cylindrical micelles. Their morphologies are dictated by their chain length, degree of hydrophilicity, and hydrophilic weight fraction of the ELP block. We find that (1) independent of the length of the corona-forming ELP block there is a minimum threshold in the length of the RLP block below which self-assembly does not occur, but that once that threshold is crossed, (2) the RLP block length is a unique molecular parameter to independently tune self-assembly and (3) increasing the hydrophobicity of the corona-forming ELP drives a transition from spherical to cylindrical morphology. Unlike the self-assembly of purely ELP-based block copolymers, the self-assembly of RLP–ELPs can be understood by simple principles of polymer physics relating hydrophilic weight fraction and polymer–polymer and polymer–solvent interactions to micellar morphology, which is important as it provides a route for the de novo design of desired nanoscale morphologies from first principles

Inducible Fibril Formation of Silk-Elastin Diblocks

Silk-elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk-elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk-elastin diblock polypeptides on fibril formation. For silk-elastin diblocks ESm-SQn, in which the elastin pentamer repeat is ES = GSGVP and the crystallizable silk octamer repeat is SQ = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks (m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation.</p

Inducible Fibril Formation of Silk–Elastin Diblocks

Silk-elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk-elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk-elastin diblock polypeptides on fibril formation. For silk-elastin diblocks ESm-SQn, in which the elastin pentamer repeat is ES = GSGVP and the crystallizable silk octamer repeat is SQ = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks (m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation.</p

Phase Behavior and Self-Assembly of Perfectly Sequence-Defined and Monodisperse Multiblock Copolypeptides

This paper investigates how the properties of multiblock copolypeptides can be tuned by their block architecture, defined by the size and distribution of blocks along the polymer chain. These parameters were explored by the precise, genetically encoded synthesis of recombinant elastin-like polypeptides (ELPs). A family of ELPs was synthesized in which the composition and length were conserved while the block length and distribution were varied, thus creating 11 ELPs with unique block architectures. To our knowledge, these polymers are unprecedented in their intricately and precisely varied architectures. ELPs exhibit lower critical solution temperature (LCST) behavior and micellar self-assembly, both of which impart easily measured physicochemical properties to the copolymers, providing insight into polymer hydrophobicity and self-assembly into higher order structures, as a function of solution temperature. Even subtle variation in block architecture changed the LCST phase behavior and morphology of these ELPs, measured by their temperature-triggered phase transition and nanoscale self-assembly. Size and morphology of polypeptide micelles could be tuned solely by controlling the block architecture, thus demonstrating that when sequence can be precisely controlled, nanoscale self-assembly of polypeptides can be modulated by block architecture

Paper Slips

Watermar

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide

Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa)132 to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP)80. The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides.</p

Nature of amorphous hydrophilic block affects self-assembly of an artificial viral coat polypeptide

Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa)132 to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP)80. The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides