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Proteins and synthetic polymers can undergo phase transitions in response to changes to intensive solution parameters such as temperature, proton chemical potentials (pH), and hydrostatic pressure. For proteins and protein-based polymers, the information required for stimulus-responsive phase transitions is encoded in their amino acid sequence. Here, we review some of the key physical principles that govern the phase transitions of archetypal intrinsically disordered protein polymers (IDPPs). These are disordered proteins with repetitive amino acid sequences. Advances in recombinant technologies have enabled the design and synthesis of protein sequences of a variety of sequence complexities and lengths. We summarize insights that have been gleaned from the design and characterization of IDPPs that undergo thermo-responsive phase transitions and build on these insights to present a general framework for IDPPs with pH and pressure responsive phase behavior. In doing so, we connect the stimulus-responsive phase behavior of IDPPs with repetitive sequences to the coil-to-globule transitions that these sequences undergo at the single-chain level in response to changes in stimuli. The proposed framework and ongoing studies of stimulus-responsive phase behavior of designed IDPPs have direct implications in bioengineering, where designing sequences with bespoke material properties broadens the spectrum of applications, and in biology and medicine for understanding the sequence-specific driving forces for the formation of protein-based membraneless organelles as well as biological matrices that act as scaffolds for cells and mediators of cell-to-cell communication.

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.

Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.

Well-defined tunable nanostructures formed through the hierarchical self-assembly of peptide building blocks have drawn significant attention due to their potential applications in biomedical science. Artificial protein polymers derived from elastin-like polypeptides (ELPs), which are based on the repeating sequence of tropoelastin (the water-soluble precursor to elastin), provide a promising platform for creating nanostructures due to their biocompatibility, ease of synthesis, and customizable architecture. By designing the sequence and composition of ELPs at the gene level, their physicochemical properties can be controlled to a degree that is unmatched by synthetic polymers. A variety of ELP-based nanostructures are designed, inspired by the self-assembly of elastin and other proteins in biological systems. The choice of building blocks determines not only the physical properties of the nanostructures, but also their self-assembly into architectures ranging from spherical micelles to elongated nanofibers. This review focuses on the molecular determinants of ELP and ELP-hybrid self-assembly and formation of spherical, rod-like, worm-like, fibrillar, and vesicle architectures. A brief discussion of the potential biomedical applications of these supramolecular assemblies is also included.

Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21. First, the ELP acts as a solubility enhancer during E. coli expression, yielding active fusion protein from the soluble cell lysate fraction and eliminating the protein refolding steps that are required for purification of FGF21 from inclusion bodies. Second, the ELP's phase transition behavior is exploited for facile chromatography-free purification of the ELP-FGF21 fusion. Third, the composition and molecular weight of the ELP are designed such that the ELP-FGF21 fusion undergoes a phase transition triggered solely by body heat, resulting in an immiscible viscous phase upon subcutaneous (s.c.) injection and thereby creating an injectable depot. Indeed, a single s.c. injection of ELP-FGF21 affords up to five days of sustained glycemic control in ob/ob mice. The ELP fusion partner massively streamlines production and purification of FGF21, while providing a controlled release method for delivery that reduces the frequency of injection, thereby enhancing the pharmacological properties of FGF21 as a protein drug to treat metabolic disease.

Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences.

In the version of this Article originally published, one of the authors' names was incorrectly given as Jeffery Schaal; it should have been Jeffrey L. Schaal. This has been corrected in all versions of the Article.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

A new class of thermally responsive protein based block co-polymers shows tunable hysteresis in their thermal transitions. The sequences encompass intrinsically disordered regions comprising of repeats of Elastin-Like Polypeptides (ELPs). These are interspersed with alpha-helical polyalanine domains. The lower critical solution temperatures (LCSTs) measured along the heating and cooling arms are tunable and can be non-overlapping. The number and type of alanine-rich blocks determine the extent of hysteresis. We have developed a phenomenological model that reproduces the experimentally observed tunable hysteresis for block copolymeric sequences. This requires an imbalance between the strengths of homotypic interactions between alanine-rich regions and ELP repeats. Additionally, the ELPs and alanine-rich regions have to be immiscible with one another. These features engender micro-phase separation whereby the block copolymers form spherical clusters comprising of alanine-rich cores and ELP coronas. Upon raising the temperature above the LCST, the clusters are drawn to one another by favorable interactions among ELPs and these clusters further network via domain swapping of alanine-rich regions. Lowering the temperature below the LCST leads to dispersion of the clusters by weakening of homotypic interactions between ELPs. However, the domain swapped states persist and this maintains the physical networking of clusters thus giving rise to hysteresis. Domain swapping and the persistence of this state below the LCST are governed by the energy gap between the homotypic interactions of ELPs vis-a-vis alanine-rich regions. Our findings have direct bearing on the de novo design of responsive materials based on IDPs, where tunable hysteresis can be used to encode memory effects, and for understanding the complexities of sequence-encoded phase behavior of archetypal low complexity disordered proteins.

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

Wilms' tumors (WTs) exhibit more than one pattern of differentiation, each of which is associated with distinctive clinical features and treatment responses. Mutations in the WT1 gene are found predominantly in WTs with stromal histology. To better understand the biological and clinical features in different WTs, we have analyzed WTs with and without WT1 mutations for a set of parameters.Twenty-two new WTs were analyzed for WT1 mutations by PCR single-strand conformational polymorphism. Five tumors with WT1 mutations and six tumors without WT1 mutations were studied for the presence of WT1 transcripts and protein as well as for the expression of differentiation markers.Two new WT1 mutations were identified in stromal-predominant tumors, and none were identified in the other histological subtypes. Tumors with WT1 mutations expressed mutant messages, cytoplasmic truncated WT1 proteins, and muscle markers. In contrast, blastemal-predominant tumors without mutations showed nuclear WT1 protein staining. Both tumor types were positive for markers of early-induced mesenchyme and one marker of uninduced mesenchyme, but blastemal-predominant tumors also expressed cytokeratin, suggesting that these are further along the epithelial differentiation pathway.Our data show that the two-hit inactivation of WT1 is operative in stromal-predominant WTs. Cells without functional nuclear WT1 protein start a faulty differentiation program. In contrast, blastemal-predominant tumors express wild-type WT1 and show early signs of epithelialization. The extensive rhabdomyomatous differentiation and the presence of WT1 mutations may be used as a diagnostic tool to identify a tumor subtype that seems to respond poorly to chemotherapy. These studies provide a foundation for improvement in tumor classification and ultimately for the development of more individualized tumor treatments.