Tamara L. Kinzer-Ursem

Selective Functionalization of the Protein N Terminus with N-Myristoyl Transferase for Bioconjugation in Cell Lysate

A site to behold: Robust site-specific functionalization of engineered proteins is achieved with N-myristoyl transferase (NMT) in bacterial cells. NMT tolerates non-natural substrate proteins as well as reactive fatty acid tags, rendering it a powerful tool for protein conjugation applications, including the construction of protein microarrays from lysate.

Physical characterization of nanoparticle size and surface modification using particle scattering diffusometry

As the field of colloidal science continues to expand, tools for rapid and accurate physiochemical characterization of colloidal particles will become increasingly important. Here, we present Particle Scattering Diffusometry (PSD), a method that utilizes dark field microscopy and the principles of particle image velocimetry to measure the diffusivity of particles undergoing Brownian motion. PSD measures the diffusion coefficient of particles as small as 30 nm in diameter and is used to characterize changes in particle size and distribution as a function of small, label-free, surface modifications of particles. We demonstrate the rapid sizing of particles using three orders-of-magnitude less sample volume than current standard techniques and use PSD to quantify particle uniformity. Furthermore, PSD is sensitive enough to detect biomolecular surface modifications of nanometer thickness. With these capabilities, PSD can reliably aid in a wide variety of applications, including colloid sizing, particle corona characterization, protein footprinting, and quantifying biomolecule activity.

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications

Calmodulin (CaM) is a widely studied Ca(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.

Optoelectric patterning: Effect of electrode material and thickness on laser-induced AC electrothermal flow

Rapid electrokinetic patterning (REP) is an emerging optoelectric technique that takes advantage of laser‐induced AC electrothermal flow and particle‐electrode interactions to trap and translate particles. The electrothermal flow in REP is driven by the temperature rise induced by the laser absorption in the thin electrode layer. In previous REP applications 350–700 nm indium tin oxide (ITO) layers have been used as electrodes. In this study, we show that ITO is an inefficient electrode choice as more than 92% of the irradiated laser on the ITO electrodes is transmitted without absorption. Using theoretical, computational, and experimental approaches, we demonstrate that for a given laser power the temperature rise is controlled by both the electrode material and its thickness. A 25‐nm thick Ti electrode creates an electrothermal flow of the same speed as a 700‐nm thick ITO electrode while requiring only 14% of the laser power used by ITO. These results represent an important step in the design of low‐cost portable REP systems by lowering the material cost and power consumption of the system.

Competitive tuning: Competition's role in setting the frequency-dependence of Ca2+-dependent proteins

A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca2+) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca2+/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca2+ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca2+/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca2+/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca2+, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca2+ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the in vivo frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca2+/CaM-dependent protein kinase II in knockout models of neurogranin. We conclude that competitive tuning could be an important dynamic process underlying synaptic plasticity.

DNA Microviscosity Characterization with Particle Diffusometry for Downstream DNA Detection Applications

Analytical characterization of DNA microviscosity provides critical biophysical insights into nuclear crowding, nucleic acid based pharmaceutical development, and nucleic acid based biosensor device design. However, most viscosity characterization methods require large sample volumes and destructive testing. In contrast, particle diffusometry permits in situ analysis of DNA microviscosity with short measurement times (8 s) using small volumes (<3 μL) which are compatible with DNA preparatory procedures. This unconventional biosensing approach involves measuring the change in sample viscosity using image processing and correlation-based algorithms. Particle diffusometry requires only a fluorescence microscope equipped with a charge-coupled device (CCD) camera and is a nondestructive measurement method. We use particle diffusometry to characterize the effect of DNA topology, length, and concentration on solution viscosity. In addition, we use particle diffusometry to detect the amplification of DNA from Staphylococcus aureus and Klebsiella pneumoniae, two pathogens commonly related to neonatal sepsis. Successful characterization of pathogen amplification with particle diffusometry provides a new opportunity to apply viscosity characterization toward downstream applications in nucleic acid based pathogen detection.

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics.

An automated workflow for quantifying RNA transcripts in individual cells in large data-sets

Graphical abstract A noisy image of fluorescently-labeled mRNA transcripts can be analyzed by Cell-by-Cell Relative Integrated Transcript (CCRIT) Quantification to automatically identify cells and cell clusters and quantify each cell’s mRNA expression level.

Relieving the Pressure on Tissue Development

Understanding how tissue patterning emerges during embryogenesis is a long-standing goal of developmental biology. In particular, the complex interplay between extracellular and intracellular mechanical forces and biochemical cues that lead to cellular differentiation and ultimately adult tissue remains an active area of investigation. One of the main challenges has been that experimental methods that allow for the simultaneous measurement of mechanical force and biochemical signaling are limited.