Engineering topics
Buchko, Garry W.
Publications and source records attributed to Buchko, Garry W..
Ameloblastin binding to biomimetic models of cell membranes – A continuum of intrinsic disorder
A 37-residue amino acid sequence corresponding to the segment encoded by exon-5 of murine ameloblastin (Ambn), AB2 (Y67-Q103), has been implicated with membrane association, ameloblastin self-assembly, and amelogenin-binding. Here, our aim was to characterize, at the residue level, the structural behavior of AB2 bound to chemical mimics of biological membranes using NMR spectroscopy. To better define the structure of AB2 using NMR-based methods, recombinant 13 C- and 15 N-labelled AB2 (*AB2) was prepared and data collected free in solution and with deuterated dodecylphosphocholine (dPC) micelles, deuterated bicelles, and both small and large unilamellar vesicles. Amide chemical shift and intensity perturbations observed in 1 H- 15 N HSQC spectra of *AB2 in the presence of bicelles and dPC micelles suggest that a region of *AB2, S6-E36 (murine Ambn S68 – E98), associates with the membrane biomimetics. A CSI-3 analysis of the NMR chemical shift assignments for *AB2 free in solution and bound to dPC micelles indicated the peptide remains disordered except for the adoption of a short, 12-residue α-helix, F10-G21 (murine Ambn F72-G83). In dPC micelles, the NOE NMR data was void of patterns characteristic of long-lived helical structure indicating this helix was transient in nature. A continuum of intrinsic disorder in the membrane-bound state may be responsible for ameloblastin’s ability to dynamically interact with multiple partners at the same site during amelogenesis.
SAXS of murine amelogenin identifies a persistent dimeric species from pH 5.0 to 8.0
Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Here, using advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at serine-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (R g = 38.4 Å) and nanospheres at pH 8.0 (R g = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0 ~ 80% of the population is a heterogenous dimeric species that increases to ~ 100% at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0 ~ 40% of the amelogenin still remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.
Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils
Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.
Resolving protein-mineral interfacial interactions during in vitro mineralization by atom probe tomography
Organic macromolecules exert remarkable control over the nucleation and growth of inorganic crystallites during (bio)mineralization, as exemplified during enamel formation where the protein amelogenin regulates the formation of hydroxyapatite (HAP). However, it is poorly understood how fundamental processes at the organic-inorganic interface, such as protein adsorption and/or incorporation into minerals, regulates nucleation and crystal growth due to technical challenges in observing and characterizing mineral-bound organics at high-resolution. Here, atom probe tomography techniques were developed and applied to characterize amelogenin-mineralized HAP particles in vitro, revealing distinct organic-inorganic interfacial structures and processes at the nanoscale. Specifically, visualization of amelogenin across the mineralized particulate demonstrates protein can become entrapped during HAP crystal aggregation and fusion. Identification of protein signatures and structural interpretations were further supported by standards analyses, i.e., defined HAP surfaces with and without amelogenin adsorbed. These findings represent a significant advance in the characterization of interfacial structures and, more so, interpretation of fundamental organic-inorganic processes and mechanisms influencing crystal growth. Ultimately, this approach can be broadly applied to inform how potentially unique and diverse organic-inorganic interactions at different stages regulates the growth and evolution of various biominerals.
Plant defensin MtDef4–derived antifungal peptide with multiple modes of action and potential as a bio–inspired fungicide
Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.
High-yield recombinant bacterial expression of 13 C-, 15 N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol
Amelogenin constitutes ~ 90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates – enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13 C- and 15 N-labelling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and a lingering question overshadowing decades of enamel research with recombinant amelogenin is the effect of phosphorylation on its biophysical properties. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13 C-, 15 N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13 C-, 15 N-labeled media and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional 31P NMR spectra, comparison of 1 H- 15 N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such 13 C-, 15 N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.
Changes in the C-terminal, N-terminal, and histidine regions of amelogenin reveal the role of oligomer quaternary structure on adsorption and hydroxyapatite mineralization
Adsorption interactions between amelogenin and calcium phosphate minerals are believed to be important to amelogenin’s function in enamel formation, however, the role of specific amino acid residues and domains within the protein in controlling adsorption is not well known. We synthesized “mechanistic probes” by systematically removing charged regions of amelogenin in order to elucidate their roles. The probes included amelogenin without the charged residues in the N-terminus (SEKR), without two, three, or eight histidines (H) in the central protein region (H2, H3, H8), or without the C-terminal residues (Delta). In-situ atomic force microscopy (AFM) adsorption studies onto hydroxyapatite (HAP) single crystals confirmed that the C-terminus was the dominant domain in promoting adsorption. We propose that subtle changes in protein-protein interactions for proteins with histidines and N-terminal residues removed resulted in changes in the oligomer quaternary size and structure that also affected protein adsorption. HAP mineralization studies revealed that the oligomer-HAP binding energy and protein layer thickness were factors in controlling the amorphous calcium phosphate (ACP) to HAP induction time. Our studies with mechanistic probes reveal the importance of the oligomer quaternary structure in controlling amelogenin adsorption and HAP mineralization.
Structural characterization of a soil viral auxiliary metabolic gene product – a functional chitosanase
Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins. We focus on AMGs that potentially encode chitosanase enzymes that metabolize chitin – a common carbon polymer. We express and functionally screen several chitosanase genes identified from environmental metagenomes. One expressed protein showing endo-chitosanase activity (V-Csn) is crystalized and structurally characterized at ultra-high resolution, thus representing the structure of a soil viral AMG product. This structure provides details about the active site, and together with structure models determined using AlphaFold, facilitates understanding of substrate specificity and enzyme mechanism. Our findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts.
Understanding Proton Movement in [Fe-Fe] Hydrogenases
Nature uses specialized metalloenzymes to carry out small molecule activation reactions, including CO 2 fixation, O 2 activation, and proton reduction, with unparalleled efficiency, rates, and selectivity. The latter reactions are performed by hydrogenases, protein metallo-complexes that interconverts H 2 to protons and electrons (H 2 oxidation) and the reverse reaction (H 2 production) with incredibly low energy input and amazingly fast kinetics. Reproducing both the activity and efficiency of metalloenzymes in sustainable anthropogenic systems remains one of the “holy grails” of inorganic chemistry. However, identifying the precise molecular components responsible for these desirable properties has been challenging in the natural metalloenzymes, hindering efforts to develop analogous processes in synthetic compounds. Considering the inherent complexity of a metalloenzyme and the many interactions, both strong and weak, that contribute to the function of an enzyme, we have elected to model natural metalloenzymes on a biochemical platform. Towards this end, we have a developed structural, functional, and mechanistic mimic of the [Ni-Fe] hydrogenases within a robust protein scaffold, rubredoxin, to understand the influence of the secondary coordination environment on the metal center. This involved preparing a series of three rubredoxin constructs containing a single point mutation at Val positions and making the NMR chemical shift assignments for the paramagnetic (nickel-substituted) and non-paramagnetic (zinc-substituted) form. These physical studies were complemented with in silico molecular dynamic studies on proteins with [Fe-S] clusters to engineer new proton channels to test in vitro . To assist our search for new natural metalloprotein scaffolds within the vast number of sequenced genomes, we created a neural network-based program to identify proteins with specific metal-binding sites. These computational and physical studies with metalloenzymes provide direct insight into the fundamental chemical principles driving the natural systems and offer design principles for developing catalysts that utilize analogous principles.
Expression and structure of the Chlamydia trachomatis DksA ortholog
Chlamydia trachomatis is a bacterial obligate intracellular parasite and a significant cause of human disease, including sexually transmitted infections and trachoma. The bacterial RNA polymerase-binding protein DksA is a transcription factor integral to the multicomponent bacterial stress response pathway known as the stringent response. The genome of C. trachomatis encodes a DksA ortholog (DksA Ct ) that is maximally expressed at 15–20 h post infection, a time frame correlating with the onset of transition between the replicative reticulate body (RB) and infectious elementary body (EB) forms of the pathogen. Ectopic overexpression of DksA Ct in C. trachomatis prior to RB–EB transitions during infection of HeLa cells resulted in a 39.3% reduction in overall replication (yield) and a 49.6% reduction in recovered EBs. While the overall domain organization of DksA Ct is similar to the DksA ortholog of Escherichia coli (DksA Ec ), DksA Ct did not functionally complement DksA Ec . Transcription of dksA Ct is regulated by tandem promoters, one of which also controls expression of nrdR, encoding a negative regulator of deoxyribonucleotide biosynthesis. The phenotype resulting from ectopic expression of DksA Ct and the correlation between dksA Ct and nrdR expression is consistent with a role for DksA Ct in the C. trachomatis developmental cycle.
Structural Characterization of Cytochrome [subscript c; beta-Met] from an Ammonia-Oxidizing Bacteri
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Controls of nature: Secondary, tertiary, and quaternary structure of the enamel protein amelogenin in solution and on hydroxyapatite
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Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection
In the indeterminate nodules of a model legume Medicago truncatula, ~700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel β-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea. In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.
Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry
Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound’s cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 μge/μL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 μM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis .
A Positive Charge in the Outer Coordination Sphere of an Artificial Enzyme Increases CO 2 Hydrogenation
The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(P Et 2 N glycine P Et 2 )2 ] – , immobilized covalently within a protein scaffold was activated for CO 2 hydrogenation. In this work, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO 2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1 H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h –1 , and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO 2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.
A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466
In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The PhenoTarget approach combines the strengths of the PDD and TDD approaches. Phenotypic screening is conducted initially to detect cellular active components and the hits are then screened against a panel of putative targets. This PhenoTarget protocol can be equally applied to pure compound libraries as well as natural product fractions. Here we described the use of the PhenoTarget approach to identify an anti-tuberculosis lead compound. Fractions from Polycarpa aurata were identified with activity against Mycobacterium tuberculosis H37Rv. Native magnetic resonance mass spectrometry (MRMS) against a panel of 37 proteins from Mycobacterium proteomes showed that a fraction from a 95% ethanol re-extraction specifically formed a protein-ligand complex with Rv1466, a putative uncharacterized Mycobacterium tuberculosis protein. The natural product responsible was isolated and characterized to be polycarpine. The molecular weight of the ligand bound to Rv1466, 233 Da, was half the molecular weight of polycarpine less one proton, indicating that polycarpine formed a covalent bond with Rv1466.