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1.  Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools 
Analytical biochemistry  2014;458:58-60.
In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300 s detection window.
PMCID: PMC4341968  PMID: 24780222
Optogenetic protein; CIB1; CIBN; CRY2; FRET; FCS
2.  Measurement of the Temperature of the Resting Rotor in Analytical Ultracentrifugation 
Analytical biochemistry  2014;458:37-39.
Accurate measurements of rotor temperature are critical for the interpretation of hydro dynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor utilizing iButton® temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2 °C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.
PMCID: PMC4080836  PMID: 24799348
sedimentation velocity; hydrodynamics; temperature calibration
3.  A Minimum Variance Method for Genome-wide Data-driven Normalization of qRT-PCR Expression Data 
Analytical biochemistry  2014;458:11-13.
Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of non-detection (~50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods.
PMCID: PMC4100614  PMID: 24780223
Multiplex qRT-PCR; Data-driven normalization
4.  Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay 
Analytical biochemistry  2014;457:74-84.
Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.
PMCID: PMC4512654  PMID: 24751466
Factor XIII; transglutaminase; coagulation; substrate specificity; kinetics; cross-linking; mass spectrometry
5.  Characterization of the Specificity of O-GlcNAc Reactive Antibodies Under Conditions of Starvation and Stress 
Analytical biochemistry  2014;457:8-18.
The dynamic modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked β-N-acetyl-D-Glucosamine (O-GlcNAc) has been shown to regulate over 3,000 proteins in a manner analogous to protein phosphorylation. O-GlcNAcylation regulates the cellular stress response, the cell cycle, and is implicated in the etiology of neurodegeneration, type II diabetes, and cancer. The antibody CTD110.6 is often used to detect changes in the O-GlcNAc modification. Recently, it has been demonstrated that CTD110.6 recognizes N-linked N,N’-diacetylchitobiose, which is thought to accumulate in cells experiencing severe glucose deprivation. In this study, we have addressed two questions: 1) Which other antibodies used to detect O-GlcNAc cross-react with N-linked N,N’-diacetylchitobiose? 2) Does N-linked N,N’-diacetylchitobiose accumulate in response to other cellular stressors? To delineate between O-GlcNAc and N-linked N,N’-diacetylchitobiose, we developed a workflow that has been used to confirm the specificity of a variety of O-GlcNAc specific antibodies. Using this workflow we demonstrated that heat shock, osmotic stress, endoplasmic reticulum stress, oxidative stress, DNA damage, proteasomal inhibition, and ATP depletion induce O-GlcNAcylation but not N-linked N,N’-diacetylchitobiose. Moreover, we demonstrated that while glucose deprivation results in an induction in both O-GlcNAc and N-linked N,N’-diacetylchitobiose, the induction of N-linked N,N’-diacetylchitobiose is exacerbated by the removal of fetal bovine serum.
PMCID: PMC4113912  PMID: 24747005
CTD110.6; O-GlcNAc; nutrient deprivation; specificity; stress-response
6.  Evaluating the effects of preanalytical variables on the stability of the human plasma proteome 
Analytical biochemistry  2015;478:14-22.
High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ≤3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.
PMCID: PMC4492164  PMID: 25769420
Plasma; Preanalytical variables; Proteomics; Mass spectrometry; Serum
7.  An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity 
Analytical biochemistry  2014;456:61-69.
Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. As these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH which is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail, and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by Co(II)-HDAC8 compared to peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-fold, 2.8-fold, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.
PMCID: PMC4470474  PMID: 24674948
histone deacetylase 8; HDAC8; acetate detection assay; fluorescence; catalysis
8.  A Differential Cell Capture Assay for Evaluating Antibody Interactions with Cell Surface Targets 
Analytical biochemistry  2010;401(2):173-181.
Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell-surface targets. Conventional binding assays such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell-surface targets on the bench top at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell-surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies’ concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.
PMCID: PMC4485450  PMID: 20178770
antibody; biotin; CD20; cell capture; ELISA; fluorescence microscopy; flow cytometry; mammalian cell; streptavidin
9.  Analysis of 3-O-sulfo group containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry 
Analytical biochemistry  2014;455:3-9.
Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetra-saccharides with 3-O-sulfo group containing glucosamine residues at their reducing ends. Quantitative on-line liquid chromatography-mass spectrometric analysis of these resistant tetrasaccharides is described. The disaccharide and tetrasaccharide composition of seven porcine intestinal heparins and five low molecular weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3 wt% of heparin and from 6.2 to 8.3 wt% of low molecular weight heparin. Since these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group containing tetrasaccharides in a heparin positively correlated (r = 0.8294) to heparin’s anticoagulant activity.
PMCID: PMC4030551  PMID: 24680753
3-O-sulfo group; tetrasaccharides; porcine intestinal heparin; anticoagulant activity; RPIP-LC-MS; heparin lyase 2
10.  Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry 
Analytical biochemistry  2014;455:35-40.
Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 µM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 µM), mid (5 µM) and high (100 µM) levels of 98.2%, 97.3% and 101.6%, respectively. Additional assay performance metrics include intra-day and inter-day coefficients of variance of < 6.4% and < 9.9%, respectively, across the range of TMAO levels. Stability studies reveal TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) shows a range of 0.73 – 126 µM, median (interquartile range) levels of 3.45 (2.25–5.79) µM, and increasing values with age. The LC/MS/MS based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic and environmental factors on TMAO levels.
PMCID: PMC4167037  PMID: 24704102
11.  Measurement of Hemoglobin Synthesis Rate in Vivo Using a Stable Isotope Method 
Analytical biochemistry  2001;291(1):118-123.
We developed a method to measure hemoglobin synthesis rate (SynHb) in humans, assuming that free glycine in the red blood cell (RBC) represents free glycine in bone marrow for hemoglobin synthesis. The present rat study examines this assumption of the method and quantifies SynHb in rats. Sprague–Dawley rats (n = 9) were studied, [2-13C]glycine was intravenously infused over 24 h (2.5 mg kg−1 h−1), blood was drawn for glycine and heme isolation, and bone marrow was harvested for glycine isolation. Isotopic enrichments of glycine and heme were measured, fractional hemoglobin synthesis rate (fSynHb% day−1) was calculated, and from this a value for SynHb (mg g−1 day−1) was derived. Mean body weight was 446 ± 10 g (mean ± SE) and hemoglobin concentration was 14 ± 0.5 g dl−1. At 24 h, the mean isotopic enrichment, atom percentage excess (APE), of the RBC free glycine (1.56 ± 0.18 APE) was similar to the bone marrow (1.68 ± 0.15 APE). The rate of incorporation of 13C into heme increased over time from 0.0004 APE/h between 6 and 12 h, to 0.0014 APE/h between 12 and 18 h, and 0.0024 APE/h between 18 and 24 h. Consequently, fSynHb (1.19 ± 0.32, 2.92 ± 0.66, and 4.22 ± 0.56% day−1, respectively) and SynHb (0.11 ± 0.03, 0.28 ± 0.05, and 0.42 ± 0.05 mg g−1 day−1, respectively) showed similar patterns over the 24-h study period. We conclude that (1) enrichment of free glycine in the circulating RBC approximates enrichment of bone marrow free glycine for heme formation and (2) this pattern of hemoglobin synthesis rate is reflecting the characteristic release and gradual maturation of reticulocytes in the circulation.
PMCID: PMC4457298  PMID: 11262164
hemoglobin synthesis; anemia; glycine; bone marrow; rats
12.  Identification of Metabolites from LC-EC Profiling: GC-MS and Re-Fractionation Provide Essential Information Orthogonal to LCMS/microNMR 
Analytical biochemistry  2014;454:23-32.
HPLC-coulometric electrode-array detection (LC-EC) is a sensitive, quantitative and robust metabolomics profiling tool that complements the commonly used MS and NMR-based approaches. However, LC-EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC-EC profiling, combined with LC-ESI-MS and microNMR. This methodology is now extended to include: (i) GC-EI-MS analysis to fill structural gaps left by LC-ESI-MS and NMR, and (ii) secondary fractionation of LC-collected fractions containing multiple co-eluting analytes. GC-EI-MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion-suppression in LC-ESI-MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples, including: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC-MS, the direct detection of carboxylic groups and sulfonic groups in 1H NMR, or non-volatile species in GC-MS).; (ii) detection of labile compounds, (iii) resolution of closely eluting and/or co-eluting compounds and, (iv) the capability to harness structural similarities common in many biologically-related, LC-EC detectable compounds.
PMCID: PMC4034759  PMID: 24657819
Metabolite characterization; Secondary Fractionation; LC-EC; LC-MS; NMR; GC-MS
13.  Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis 
Analytical biochemistry  2014;454:44-52.
Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE). Gels are run at a low, constant voltage (~ 10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent upon the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20–25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10 – 100 bp): high voltages and short run times produced sharper bands and higher resolution.
PMCID: PMC4021863  PMID: 24637158
gel electrophoresis; agarose; nucleic acids; current; resolution
14.  Dual Genetically Encoded Phage-Displayed Ligands 
Analytical biochemistry  2014;453:1-3.
M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different, genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display.
PMCID: PMC4024456  PMID: 24607794
15.  Microplate-based screening for small molecule inhibitors of Neuropilin-2/VEGF-C interactions 
Analytical biochemistry  2014;453:4-6.
Vascular Endothelial Growth Factor-C (VEGF-C) is a secreted growth factor essential for lymphangiogenesis. VEGF-C functions in both physiological and pathological lymphangiogenesis, particularly in tumor metastasis, making it an attractive therapeutic target. Members of two families of cell surface receptors transduce VEGF-C signals, Neuropilin-2 (Nrp2) and VEGF-receptor (VEGFR)-2/3. Nrp2 is a promising target for inhibition since it is highly expressed in lymphatic vessels. Here we describe a microplate-based assay for discovery of VEGF-C/Nrp2 inhibitors. We optimize this assay for use in screening an inhibitor library and identify three novel Nrp2/VEGF-C binding inhibitors from the NIH Clinical Collection small molecule library.
PMCID: PMC4036672  PMID: 24583243
Neuropilin; VEGF; ligand; receptor; assay; inhibitor
16.  Development of a fluorescence resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity 
Analytical biochemistry  2014;453:61-69.
Due to their efficiency in the hydrolysis of the collagen triple-helix, Clostridial histolyticum collagenases are utilized for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of Clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Föster resonance energy transfer (FRET) triple-helical peptide (fTHP) substrate has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence Gly-mep-Flp-(Gly-Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] had a melting temperature (Tm) of 36.2 °C and was hydrolyzed efficiently by bacterial collagenase (kcat/KM = 25,000 sec−1M−1), but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity towards triple-helices and thus potential application for evaluating the efficiency of cell isolation by collagenases.
PMCID: PMC4260936  PMID: 24608089
bacterial collagenase; clostripain; collagen; FRET protease assay; islet isolation; stem cell isolation
17.  Extraction of RNA from Ca-Alginate Encapsulated Yeast for Transcriptional Profiling 
Analytical biochemistry  2009;391(2):160-162.
We have developed a method for preparing high-quality total RNA from Ca-alginate encapsulated Saccharomyces cerevisiae that is suitable for microarray analysis. Encapsulated cells were harvested from immobilized cell reactors and flash-frozen in liquid nitrogen. Following low-temperature mechanical disruption, cells were freed from Ca-alginate by reverse ionotropic gelation, purified by centrifugation, and total RNA was extracted using hot acid phenol. The yield and quality of the RNA were consistently high; the RNA was free of contaminating alginate, and in microarray analysis performed as well as RNA isolated from planktonic cells.
PMCID: PMC4426872  PMID: 19406096
Alginate-encapsulated yeasts; RNA isolation; Expression profiling
18.  Enhanced assay of endothelial exocytosis using extracellular matrix components 
Analytical biochemistry  2014;452:19-24.
Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing pro-thrombotic and pro-inflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I coated plates and cultured in the confluent state for 7–12 days in low serum media showed robust secretion of von Willebrand Factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening.
PMCID: PMC4048756  PMID: 24561025
endothelial; exocytosis; VWF; extracellular matrix
19.  Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation 
Analytical biochemistry  2014;451:25-27.
We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay utilizes 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins including most GPCRs.
PMCID: PMC3969759  PMID: 24463015
Palmitoylation; click chemistry; GPCRs; mu-opioid receptor; immunoprecipitation; Western blotting
20.  Improved Measurement of the Rotor Temperature in Analytical Ultracentrifugation 
Analytical biochemistry  2014;451:69-75.
Sedimentation velocity is a classical method for measuring the hydrodynamic, translational friction coefficient of biological macromolecules. In a recent study, comparing various analytical ultracentrifuges, we have shown that external calibration of the scan time, radial magnification, and temperature are critically important for accurate measurements (Anal. Biochem., 2013, doi: 10.1016/j.ab.2013.05.011). To achieve accurate temperature calibration, we have introduced the use of an autonomous miniature temperature logging integrated circuit (Maxim Thermochron iButton ™) that can be inserted in an ultracentrifugation cell assembly and spun at low rotor speeds. In the present work, we developed an improved holder for the temperature sensor located in the rotor handle. This has the advantage of not reducing the rotor capacity and allows for a direct temperature measurement of the spinning rotor during high-speed sedimentation velocity experiments up to 60,000 rpm. We demonstrate the sensitivity of this approach by monitoring the adiabatic cooling due to rotor stretching during rotor acceleration, and the reverse process upon rotor deceleration. Based on this, we developed a procedure to approximate isothermal rotor acceleration for better temperature control.
PMCID: PMC4051143  PMID: 24530285
Sedimentation velocity; temperature calibration
21.  A Sensitive Mass-spectrum Assay to Characterize Engineered Methionine Adenosyltransferases with S-Alkyl Methionine Analogues as Substrates 
Analytical biochemistry  2013;450:11-19.
Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-L-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAM) into the SAM analogues with engineered MATs. Here we describe a general, sensitive activity assay for engineered MATs by converting the reaction products into S-alkyl-thioadenosines, followed by HPLC/MS/MS quantification. With this assay, 40 human MAT mutants were evaluated against seven SAAM as potential substrates. The structure-activity-relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity towards the bulky SAAM stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAM.
PMCID: PMC3947680  PMID: 24374249
epigenetics; methyltransferase; MAT; S-adenosyl-L-methionine; LC-MS/MS
22.  The Use of Reaction Timecourses to Determine the Level of Minor Contaminants in Enzyme Preparations† 
Analytical biochemistry  2014;450:20-26.
Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double-mutant of orotidine 5’-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093 mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.
PMCID: PMC3967724  PMID: 24393696
Enzyme Purity; Enzyme Kinetics
23.  Generating NMR Chemical Shift Assignments of Intrinsically Disordered Proteins Using Carbon-Detect NMR Methods 
Analytical biochemistry  2013;449:17-25.
There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor 1H-amide chemical shift dispersion typically observed in their spectra. Recently, 13C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multi-dimensional experiments based on the 15N,13C-CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two 3D experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy-chain connections to be built along the IDP backbone, facilitated by acquisition of amino-acid specific 15N,13C-CON detected experiments. Assignments are completed through carbon-detected, TOCSY based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through the web.
PMCID: PMC3944900  PMID: 24333248
intrinsically disordered proteins; carbon detect NMR; amino-acid filtered NMR; CAS-NMR; Pdx1
24.  Un-induced high-yield bacterial expression of fluorescent proteins 
Analytical biochemistry  2013;449:155-157.
Here we introduce a fast, cost-effective, and highly efficient method for production of soluble fluorescent proteins from bacteria. The method does not require optimization, and does not utilize IPTG induction. The method relies on un-induced expression in the BL21-gold (DE3) strain of E. coli and yields large amounts (up to 0.4 μmoles) of fluorescent protein from a 250 mL culture. This method is much simpler than published methods, and can be used to produce any fluorescent protein that is needed in biomedical research.
PMCID: PMC3957324  PMID: 24378720
Fluorescent protein; gene expression; protein production; high-yield; FRET; His-Tag; E. coli.
25.  Glycosylation of A Disintegrin And Metalloprotease 17 (ADAM17) Affects its Activity and Inhibition 
Analytical biochemistry  2013;449:68-75.
ADAM17 is believed to be a tractable target in various diseases including cancer and rheumatoid arthritis; however, it is not known whether glycosylation of ADAM17 expressed in healthy cells differs from the one found in a diseased tissue and, if so, whether glycosylation affects inhibitor binding. We expressed human ADAM17 in mammalian and insect cells and compared their glycosylation, substrate kinetics, and inhibition profiles. We found that ADAM17 expressed in mammalian cells was more heavily glycosylated than its insect-expressed analog. To determine whether differential glycosylation modulates enzymatic activity, we performed kinetic studies with both ADAM17 analogs and various TNFα-based substrates. The mammalian form of ADAM17 exhibited 10–30 fold lower kcat values than the insect analog, while the KM was unaffected, suggesting that glycosylation of ADAM17 can potentially play role in the regulating enzyme activity in vivo. Finally, we tested ADAM17 forms for inhibition by several well-characterized inhibitors. Active site zinc-binding small molecules did not exhibit differences between the two ADAM17 analogs, while a non-zinc-binding exosite inhibitor of ADAM17 showed significantly lower potency towards the mammalian-expressed analog. These results suggest that glycosylation of ADAM17 can affect cell signaling in disease and might provide opportunities for therapeutic intervention using exosite inhibitors.
PMCID: PMC4334441  PMID: 24361716
ADAM17; metalloprotease; exosites; glycosylation

Results 1-25 (664)