A project-oriented biochemistry laboratory for protein engineering and structure-function using small laccase enzyme from Streptomyces coelicolor.

An understanding of structure-function relationships in proteins is essential for modern biochemical studies. The integration of common freely accessible bioinformatics tools available online with the knowledge of protein-engineering tools provide a fundamental understanding of the application of protein structure-function for biochemical research. In order for students to apply their prior knowledge of recombinant protein technology into the understanding of protein structure-function relationships, we developed a semester-long project-oriented biochemistry laboratory experience that is the second laboratory course of a series. For easier integration of knowledge and application, we organized this course into four sequential modules: protein structure visualization/modification, mutagenesis target identification, site-directed mutagenesis, and mutant protein expression, purification, and characterization. These tasks were performed on the protein small laccase (SLAC) that was cloned and characterized by students in the previous semester during the first biochemistry laboratory course of the series. This goal-oriented project-based approach helped students apply their prior knowledge to newly introduced techniques to understand protein structure-function relationships in this research-like laboratory setting. A student assessment before and after the course demonstrated an overall increase in learning and enthusiasm for this topic.

Computational and experimental analyses of alanine racemase suggest new avenues for developing allosteric small-molecule antibiotics.

Given the ever-present threat of antibacterial resistance, there is an urgent need to identify new antibacterial drugs and targets. One such target is alanine racemase (Alr), an enzyme required for bacterial cell-wall biosynthesis. Alr is an attractive drug target because it is essential for bacterial survival but is absent in humans. Existing drugs targeting Alr lack specificity and have severe side effects. We here investigate alternative mechanisms of Alr inhibition. Alr functions exclusively as an obligate homodimer, so we probed seven conserved interactions on the dimer interface, distant from the enzymatic active site, to identify possible allosteric influences on activity. Using the Alr from Mycobacterium tuberculosis (MT) as a model, we found that the Lys261/Asp135 salt bridge is critical for catalytic activity. The Lys261Ala mutation completely inactivated the enzyme, and the Asp135Ala mutation reduced catalytic activity eight-fold. Further investigation suggested a potential drug-binding site near the Lys261/Asp135 salt bridge that may be useful for allosteric drug discovery.

Phage vs. Phage: Direct Selections of Sandwich Binding Pairs.

The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers' control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide-peptide and one Fab-peptide sandwich for the cancer and Parkinson's disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays.

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.

Single-molecule Taq DNA polymerase dynamics.

Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-µs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.

Substitution of the Methionine Axial Ligand of the T1 Copper for the Fungal-like Phenylalanine Ligand (M298F) Causes Local Structural Perturbations that Lead to Thermal Instability and Reduced Catalytic Efficiency of the Small Laccase from Streptomyces coelicolor A3(2).

Many industrial processes operate at elevated temperatures or within broad pH and salinity ranges. However, the utilization of enzymes to carry out biocatalysis in such processes is often impractical or even impossible. Laccases (EC 1.10.3.2), which constitute a large family of multicopper oxidases, have long been used in the industrial setting. Although fungal laccases are in many respects considered superior to their bacterial counterparts, the bacterial laccases have been receiving greater attention recently. Albeit lower in redox potential than fungal laccases, bacterial laccases are commonly thermally more stable, act within broader pH ranges, do not contain posttranslational modifications, and could therefore serve as a high potential scaffold for directed evolution for the production of enzymes with enhanced properties. Several examples focusing on the axial ligand mutations of the T1 copper site have been published in the past. However, structural evidence on the local and global changes induced by those mutations have thus far been of computational nature only. In this study, we set out to structurally and kinetically characterize a few of the most commonly reported axial ligand mutations of a bacterial small laccase (SLAC) from Streptomyces coelicolor. While one of the mutations (Met to Leu) equips the enzyme with better thermal stability, the other (Met to Phe) induces an opposite effect. These mutations cause local structural rearrangement of the T1 site as demonstrated by X-ray crystallography. Our analysis confirms past findings that for SLACs, single point mutations that change the identity of the axial ligand of the T1 copper are not enough to provide a substantial increase in the catalytic efficiency but can in some cases have a detrimental effect on the enzyme's thermal stability parameters instead.

Predicting COVID-19 Severity with a Specific Nucleocapsid Antibody plus Disease Risk Factor Score.

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.

Predicting COVID-19 Severity with a Specific Nucleocapsid Antibody plus Disease Risk Factor Score.

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within six days post-symptom onset and sometimes within one day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patients comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 Likelihood Ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.

Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

3D-Printed Labware for High-Throughput Immobilization of Enzymes.

In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures. Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme's loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist's time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.

Photostable and Proteolysis-Resistant Förster Resonance Energy Transfer-Based Calcium Biosensor.

Molecular sensors from protein engineering offer new methods to sensitively bind to and detect target analytes for a wide range of applications. For example, these sensors can be integrated into probes for implantation, and then yield new and valuable physiological information. Here, a new Förster resonance energy transfer (FRET)-based sensor is integrated with an optical fiber to yield a device measuring free Ca2+. This membrane encapsulated optical fiber (MEOF) device is composed of a sensor matrix that fills poly(tetrafluoroethylene) (PTFE) with an engineered troponin C (TnC) protein fused to a pair of FRET fluorophores. The FRET efficiency is modulated upon Ca2+ ion binding. The probe further comprises a second, size-excluding filter membrane that is synthesized by filling the pores of a PTFE matrix with a poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel; this design ensures protection from circulating proteases and the foreign body response. The two membranes are stacked and placed on a thin, silica optical fiber for optical excitation and detection. Results show the biosensor responds to changes in Ca2+ concentration within minutes with a sensitivity ranging from 0.01 to 10 mM Ca2+, allowing discrimination of hyper- and hypocalcemia. Furthermore, the system reversibly binds Ca2+ to allow continuous monitoring. This work paves the way for the use of engineered structure-switching proteins for continuous optical monitoring in a large number of applications.

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 pM in One Minute.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species.

Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture.

Streptomyces, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of Streptomyces coelicolor A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of S. coelicolor due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from S. coelicolor was also heterologously cloned and expressed in Escherichia coli for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, kcat/KM, of 2.0 ± 0.1 mM-1 s-1. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, kcat/KM, of 1.9 ± 0.1 mM-1 s-1.

Electrochemical Quantification of Glycated and Non-glycated Human Serum Albumin in Synthetic Urine.

A polymer-based electrode capable of specific detection of human serum albumin, and its glycated derivatives, is described. The sensor is constructed from a glass microscope slide coated with a synthesized, polythiophene film bearing a protected, iminodiacetic acid motif. The electrode surface is then further elaborated to a functional biosensor through deprotection of the iminodiacetic acid, followed by metal-affinity immobilization of a specific and high-affinity, albumin ligand. Albumin was then quantified in buffer and synthetic urine via electrochemical impedance spectroscopy. Glycated albumin was next bound to a boronic acid-modified, single-cysteine dihydrofolate reductase variant to quantify glycation ratios by square-wave voltammetry. The platform offers high sensitivity, specificity, and reproducibility in an inexpensive arrangement. The detection limits exceed the requirements for intermediate-term glycemic control monitoring in diabetes patients at 5 and 1 nM for albumin and its glycated forms, respectively.

Continuous flow biocatalysis.

The continuous flow synthesis of active pharmaceutical ingredients, value-added chemicals, and materials has grown tremendously over the past ten years. This revolution in chemical manufacturing has resulted from innovations in both new methodology and technology. This field, however, has been predominantly focused on synthetic organic chemistry, and the use of biocatalysts in continuous flow systems is only now becoming popular. Although immobilized enzymes and whole cells in batch systems are common, their continuous flow counterparts have grown rapidly over the past two years. With continuous flow systems offering improved mixing, mass transfer, thermal control, pressurized processing, decreased variation, automation, process analytical technology, and in-line purification, the combination of biocatalysis and flow chemistry opens powerful new process windows. This Review explores continuous flow biocatalysts with emphasis on new technology, enzymes, whole cells, co-factor recycling, and immobilization methods for the synthesis of pharmaceuticals, value-added chemicals, and materials.

Directed evolution and biophysical characterization of a full-length, soluble, human caveolin-1 variant.

Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cavsol, for expression in E. coli. Cavsol folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and the polymerase I and transcript release factor. As expected, cavsol does not bind off-target proteins. Cellular studies show that cavsol retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cavsol forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cavsol's secondary structure is a mixture of α-helices and ß-strands. Isothermal titration calorimetry experiments reveal that cavsol binds to gp41 and PKA with low micromolar binding affinity (KD). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins.

Small laccase from streptomyces coelicolor-an ideal model protein/enzyme for undergraduate laboratory experience.

A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students perform the tasks of cloning, expression, purification, analysis, and characterization of small laccase (SLAC) from Streptomyces coelicolor. SLAC is an extremely robust well-characterized protein/enzyme, which serves as an ideal model for undergraduate teaching laboratories. Also, this goal-oriented research-like approach helps students to unite the concepts of biochemistry and molecular biology and appreciate the utility of the methods more effectively. A student assessment before and after the course demonstrated an overall increase in learning and enthusiasm on the topic of modern protein chemistry. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):172-181, 2018.