Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin.

Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 µmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.

Scalable Production of Peptides for Enhanced Struvite Formation via Expression on the Surface of Genetically Engineered Microbes.

A promising method for recycling phosphate from wastewater is through precipitation of struvite (MgNH4PO4·6H2O), a slow-release fertilizer. Peptides have been shown to increase the yield of struvite formation, but producing peptides via solid phase synthesis is cost prohibitive. This work investigates the effects of peptide-expressing bacteria on struvite precipitation to provide a sustainable and cost-efficient means to enhance struvite precipitation. A peptide known for increased struvite yield was expressed on a membrane protein in Escherichia coli(E. coli), and then 5 mL precipitation reactions were performed in 50 mL culture tubes for at least 15 min. The yield of struvite crystals was examined, with the presence of peptide-expressing E. coli inducing significantly higher yields than nonpeptide-expressing E. coli when normalized to the amount of bacteria. The precipitate was identified as struvite through Fourier transform infrared spectroscopy and energy dispersive spectroscopy, while the morphology and size of the crystals were analyzed through optical microscopy and scanning electron microscopy. Crystals were found to have a larger area when precipitated with the peptide-expressing bacteria. Additionally, bacteria-struvite samples were thermogravimetrically analyzed to quantify their purity and determine their thermal decomposition behavior. Overall, this study presents the benefits of a novel, microbe-driven method of struvite precipitation, offering a means for scalable implementation.

Clickable polymer scaffolds enable Ce recovery with peptide ligands.

Rare earth elements (REEs) are a vital part of many technologies with particular importance to the renewable energy sector and there is a pressing need for environmentally friendly and sustainable processes to recover and recycle them from waste streams. Functionalized polymer scaffolds are a promising means to recover REEs due to the ability to engineer both transport properties of the porous material and specificity for target ions. In this work, REE adsorbing polymer scaffolds were synthesized by first introducing poly(glycidyl methacrylate) (GMA) brushes onto porous polyvinylidene fluoride (PVDF) surface through activator generated electron transfer atom transfer radical polymerization (AGET ATRP). Azide moieties were then introduced through a ring opening reaction of GMA. Subsequently, REE-binding peptides were conjugated to the polymer surface through copper catalyzed azide alkyne cycloaddition (CuAAC) click chemistry. The presence of GMA, azide, and peptide was confirmed through Fourier transform infrared spectroscopy. Polymer scaffolds functionalized with the REE-binding peptide bound cerium, while polymer scaffolds functionalized with a scrambled control peptide bound significantly less cerium. Importantly, this study shows that the REE binding peptide retains its functionality when bound to a polymer surface. The conjugation strategy employed in this work can be used to introduce peptides onto other polymeric surfaces and tailor surface specificity for a wide variety of ions and small molecules.

Grafting of short elastin-like peptides using an electric field.

Surface-grafted elastin has found a wide range of uses such as sensing, tissue engineering and capture/release applications because of its ability to undergo stimuli-responsive phase transition. While various methods exist to control surface grafting in general, it is still difficult to control orientation as attachment occurs. This study investigates using an electric field as a new approach to control the surface-grafting of short elastin-like polypeptide (ELP). Characterization of ELP grafting to gold via quartz crystal microbalance with dissipation, atomic force microscopy and temperature ramping experiments revealed that the charge/hydrophobicity of the peptides, rearrangement kinetics and an applied electric field impacted the grafted morphology of ELP. Specifically, an ELP with a negative charge on the opposite end of the surface-binding moiety assembled in a more upright orientation, and a sufficient electric field pushed the charge away from the surface compared to when the same peptide was assembled in no electric field. In addition, this study demonstrated that assembling charged ELP in an applied electric field impacts transition behavior. Overall, this study reveals new strategies for achieving desirable and predictable surface properties of surface-bound ELP.

Self-Assembly and Rearrangement of a Polyproline II Helix Peptide on Gold.

Polyproline peptide sequences have gained popularity as anchors for peptide-based self-assembled monolayers (SAMs) due to their attractive properties. In this work, peptides containing the polyproline II helix (PPII) conformation were designed and assembled on gold (Au). A quartz crystal microbalance with dissipation was used to characterize SAM formation kinetics and related properties. Peptides were designed with the sequence (GPPPPPG)2C. It was discovered that a biexponential adsorption and rearrangement model describes the binding kinetics of the PPII-containing peptide on Au. In this model, an initial reversible binding step is followed by an irreversible rearrangement step, given by parameter kt. This study found kt to be approximately 0.00064 s-1 for the PPII-containing peptides. Similarly, we found that the adsorption of the PPII-containing peptide on Au, given by ΔGads, was thermodynamically favorable (-7.8 kcal mol-1) and comparable to other common thiol terminated SAMs on Au. Furthermore, we characterized SAM properties via QCM-D, Fourier-transform infrared (FTIR) spectroscopy, and electrochemical techniques to reveal high molecular density SAMs consisting of PPII helices. In addition, these SAMs were found to have high antifouling properties. Overall, this study characterizes the fundamental assembly mechanisms, particularly, rearrangement of PPII-containing peptides for the first time, which will be useful in the designing of future peptide-based SAMs with high surface coverage and antifouling properties.

Exploring the effect of a peptide additive on struvite formation and morphology: a high-throughput method.

Precipitation of struvite (MgNH4PO4·6H2O), a slow-release fertilizer, provides a means of recycling phosphate from wastewater streams. In this work, a high-throughput struvite precipitation method is developed to investigate the effects of a peptide additive. The reactions occurred in small volumes (300 µL or less) in a 96-well plate for 45 minutes. The formation of struvite was monitored by fitting absorbance at 600 nm over time to a first order model with induction time, with the addition of peptide inducing significant changes to the yield parameter and formation constant in that model. The impact of struvite seed dosing was also investigated, highlighting the importance of optimization when peptide is present. The composition of the precipitate was confirmed through Fourier-transform infrared spectroscopy, while morphology and crystal size were analyzed through optical microscopy. Crystals had a higher aspect ratio when precipitated with the peptide. Finally, the utility of the high-throughput platform was demonstrated with a 25 full factorial design to capture the effects and interactions of: magnesium dose, mixing time, seed dose, pH, and temperature. Overall, this study quantifies novel effects of a sequence-defined peptide on struvite formation and morphology via a newly developed high throughput platform.