Infrared Photodissociation Spectroscopy of Water-Tagged Ions with a Widely Tunable Quantum Cascade Laser for Planetary Science Applications.

This work presents a benchtop method for collecting the room temperature gas phase infrared (IR) action spectra of protonated amino acids and their isomers. The adopted setup uses a minimally modified commercial electrospray ionization linear ion trap mass spectrometer (ESI-LIT-MS) coupled to a broadband continuous wave (cw) quantum cascade laser (QCL) source. This approach leverages messenger assisted action spectroscopic techniques using water-tagged molecular ions with complex formation, irradiation, and subsequent analysis, all taking place within a single linear ion trap stage. This configuration thus circumvents the use of multiple mass selection and analysis stages, cryogenic buffer cells, and complex high-power laser systems typically called upon to execute these techniques. The benchtop action spectrometer is used to collect the 935-1600 cm-1 (6.2-10.7 µm) IR action spectrum of a collection of amino acids and a dipeptide with results cross referenced against literature examples obtained with a free electron laser source. Recorded IR spectra are used for the analysis of binary mixture samples composed of constitutional isomers α-alanine and ß-alanine with ratios determined to ∼4% measurement uncertainty without the aid of a front-end separation stage. This turn-key QCL-based approach is a major step in showing the viability of tag-based action spectroscopic techniques for use in future in situ planetary science sensors and general analytical applications.

Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered Peptide Tag.

Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.

Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide.

Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.