Allosteric actuation of inverse phase transition of a stimulus-responsive fusion polypeptide by ligand binding.
ABSTRACT: We report herein a biopolymer actuator with a modular design that allosterically transduces ligand binding into an aqueous demixing phase transition. The biopolymer actuator consists of two modular domains: a ligand binding protein domain, calmodulin (CaM), that is fused to a transducer domain, a stimulus-responsive elastin-like polypeptide (ELP) that exhibits a reversible lower critical solution temperature (LCST) phase transition. We demonstrate that binding of calcium to CaM spontaneously triggers the phase transition of the attached ELP, leading to formation of meso-microscale particles depending on the chain length of the ELP. This behavior is reversible as chelation of the bound calcium results in dissolution of the assembled particles, is selective for calcium as opposed to magnesium, and is abolished by the binding of a peptide ligand that is specific to calcium-bound CaM. These results are, to our knowledge, the first demonstration of biomolecular recognition-triggered, allosteric regulation of the LCST phase transition of a polymer and are significant because they expand the available triggers of the LCST transition of stimulus-responsive polymers to biochemical ligand binding. The ability to allosterically trigger the LCST transition of ELPs by biomolecular recognition will be useful for developing "smart" polymer actuators that capitalize upon the myriad ligand-protein pairs that are available from biology and for application in the design of selective pull-down assays in proteomics, drug delivery, and nanoscale biomolecular devices.
Project description:The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent β-turn structures. Labeling with Dox further enhances β-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets.
Project description:Elastin-like polypeptides (ELPs) are recombinant protein domains exhibiting lower critical solution temperature (LCST) behavior. This LCST behavior is controlled not only by intrinsic factors including amino acid composition and polypeptide chain length but also by non-ELP fusion domains. Here, we report that the presence of a composite non-ELP sequence that includes both His and T7 tags or a short Ser-Lys-Gly-Pro-Gly (SKGPG) sequence can dramatically change the LCST behavior of a positively-charged ELP domain. Both the His and T7 tags have been widely used in recombinant protein design to enable affinity chromatography and serve as epitopes for protein detection. The SKGPG sequence has been used to improve the expression of ELPs. Both the composite tag and the SKGPG sequence are <15% of the total length of the ELP fusion proteins. Despite the small size of the composite tag, its incorporation imparted pH-sensitive LCST behavior to the positively-charged ELP fusion protein. This pH sensitivity was not observed with the incorporation of the SKGPG sequence. The pH sensitivity results from both electrostatic and hydrophobic interactions between the composite tag and the positively-charged ELP domain. The hydrophobicity of the composite tag also alters the ELP interaction with Hofmeister salts by changing the overall hydrophobicity of the fusion protein. Our results suggest that incorporation of short tag sequences should be considered when designing temperature-responsive ELPs and provide insights into utilizing both electrostatic and hydrophobic interactions to design temperature-responsive recombinant proteins as well as synthetic polymers.
Project description:The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G(3)-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V(5)-120. It was also found that larger anions could bind with ELP V5A2G(3)-120 more readily in comparison with ELP V(5)-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.
Project description:This study evaluated a biodegradable drug delivery system for local cancer radiotherapy consisting of a thermally sensitive elastin-like polypeptide (ELP) conjugated to a therapeutic radionuclide. Two ELPs (49 kDa) were synthesized using genetic engineering to test the hypothesis that injectable biopolymeric depots can retain radionuclides locally and reduce the growth of tumors. A thermally sensitive polypeptide, ELP(1), was designed to spontaneously undergo a soluble-insoluble phase transition (forming viscous microparticles) between room temperature and body temperature upon intratumoral injection, while ELP(2) was designed to remain soluble upon injection and to serve as a negative control for the effect of aggregate assembly. After intratumoral administration of radionuclide conjugates of ELPs into implanted tumor xenografts in nude mice, their retention within the tumor, spatio-temporal distribution, and therapeutic effect were quantified. The residence time of the radionuclide-ELP(1) in the tumor was significantly longer than the thermally insensitive ELP(2) conjugate. In addition, the thermal transition of ELP(1) significantly protected the conjugated radionuclide from dehalogenation, whereas the conjugated radionuclide on ELP(2) was quickly eliminated from the tumor and cleaved from the biopolymer. These attributes of the thermally sensitive ELP(1) depot improved the antitumor efficacy of iodine-131 compared to the soluble ELP(2) control. This novel injectable and biodegradable depot has the potential to control advanced-stage cancers by reducing the bulk of inoperable tumors, enabling surgical removal of de-bulked tumors, and preserving healthy tissues.
Project description:Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca(2+))4-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR(11975-1999) or hRyR1(3614-3643). Both CaM and its C-domain associated in a calcium-independent manner with hRyR1(3614-3643) while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR1(11975-1999) was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR1(3614-3643) at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR(11975-1999) alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.
Project description:In this contribution, the temperature-dependent swelling behavior of vapor-deposited smart polymer thin films is shown to depend on cross-linking and deposited film thickness. Smart polymers find application in sensor and actuator setups and are mostly fabricated on delicate substrates with complex nanostructures that need to be conformally coated. As initiated chemical vapor deposition (iCVD) meets these specific requirements, the present work concentrates on temperature-dependent swelling behavior of iCVD poly(N-isopropylacrylamide) thin films. The transition between swollen and shrunken state and the corresponding lower critical solution temperature (LCST) was investigated by spectroscopic ellipsometry in water. The films' density in the dry state evaluated from X-ray reflectivity could be successfully correlated to the position of the LCST in water and was found to vary between 1.1 and 1.3 g/cm3 in the thickness range 30-330 nm. This work emphasizes the importance of insights in both the deposition process and mechanisms during swelling of smart polymeric structures.
Project description:Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.
Project description:Treatment of glioblastoma is complicated by the tumors' high resistance to chemotherapy, poor penetration of drugs across the blood brain barrier, and damaging effects of chemotherapy and radiation to normal neural tissue. To overcome these limitations, a thermally responsive polypeptide was developed for targeted delivery of therapeutic peptides to brain tumors using focused hyperthermia. The peptide carrier is based on elastin-like polypeptide (ELP), which is a thermally responsive biopolymer that forms aggregates above a characteristic transition temperature. ELP was modified with cell penetrating peptides (CPPs) to enhance delivery to brain tumors and mediate uptake across the tumor cells' plasma membranes and with a peptide inhibitor of c-Myc (H1). In rats with intracerebral gliomas, brain tumor targeting of ELP following systemic administration was enhanced up to 5-fold by the use of CPPs. When the lead CPP-ELP-fused c-Myc inhibitor was combined with focused hyperthermia of the tumors, an additional 3 fold increase in tumor polypeptide levels was observed, and 80% reduction in tumor volume, delayed onset of tumor-associated neurological deficits, and at least doubled median survival time including complete regression in 80% of animals was achieved. This work demonstrates that a c-Myc inhibitory peptide can be effectively delivered to brain tumors.
Project description:Calmodulin (CaM) is a 16.8-kDa calcium-binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix-loop-helix calcium-binding motif. CaM is composed of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3-A structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single-wavelength anomalous diffraction phasing of a selenomethionyl N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-calmodulin (apo-CaM), despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a cross-linked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggest that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either stepwise conformational transition or flexible, dynamic energetic state sampling-based transition.
Project description:The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.