15N-labeled Protein Research Articles

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.

Nanotube Array Method for Studying Lipid-Induced Conformational Changes of a Membrane Protein by Solid-State NMR

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly 15N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins.

Defining the potential of aglycone modifications for affinity/selectivity enhancement against medically relevant lectins: synthesis, activity screening, and HSQC-based NMR analysis.

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective β-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.

Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii

BackgroundThe length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii.ResultsTo achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time.ConclusionWe describe a more rapid and non-radioactive method to measure free amino acid and protein turnover. Our approach is applicable for determination of protein turnover for various proteins, which will lead to a better understanding of the relationship between protein lifetime and functionality.

Expression and purification of the aortic amyloid polypeptide medin

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer’s amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on 13C- and 15N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly 13C,15N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first 13C and 15N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins

Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z’s allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at “optimal” relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.

New stable isotope method to measure protein digestibility and response to pancreatic enzyme intake in cystic fibrosis

Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6hby adding (15)N-labeled spirulina protein and L-[ring-(2)H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [(15)N]PHE to [(2)H5]PHE. Pancreatic enzymes were ingested after 2h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-(13)C-5,5-(2)H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P<0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100min and maintained for 80min. Stratification into CF children (n=10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. Registration ClinicalTrials.gov=NCT01494909.

Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy

Two-dimensional 15N chemical shift/1H chemical shift and three-dimensional 1H–15N dipolar coupling/15N chemical shift/1H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from 1H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly 15N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear 1H–15N dipolar coupling frequency dimension is shown to select among 15N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

Stabilization of Branched Oligosaccharides: Lewisx Benefits from a Nonconventional C–H···O Hydrogen Bond

Although animal lectins usually show a high degree of specificity for glycan structures, their single-site binding affinities are typically weak, a drawback which is often compensated in biological systems by an oligovalent presentation of carbohydrate epitopes. For the design of monovalent glycomimetics, structural information regarding solution and bound conformation of the carbohydrate lead represents a valuable starting point. In this paper, we focus on the conformation of the trisaccharide Le(x) (Gal[Fucα(1-3)]β(1-4)GlcNAc). Mainly because of the unfavorable tumbling regime, the elucidation of the solution conformation of Le(x) by NMR has only been partially successful so far. Le(x) was therefore attached to a (13)C,(15)N-labeled protein. (13)C,(15)N-filtered NOESY NMR techniques at ultrahigh field allowed increasing the maximal NOE enhancement, resulting in a high number of distance restraints per glycosidic bond and, consequently, a well-defined structure. In addition to the known contributors to the conformational restriction of the Le(x) structure (exoanomeric effect, steric compression induced by the NHAc group adjacent to the linking position of L-fucose, and the hydrophobic interaction of L-fucose with the β-face of D-galactose), a nonconventional C-H···O hydrogen bond between H-C(5) of L-fucose and O(5) of D-galactose was identified. According to quantum mechanical calculations, this C-H···O hydrogen bond is the most prominent factor in stabilization, contributing 40% of the total stabilization energy. We therefore propose that the nonconventional hydrogen bond contributing to a reduction of the conformational flexibility of the Le(x) core represents a novel element of the glycocode. Its relevance to the stabilization of related branched oligosaccharides is currently being studied.

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover.

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹⁵N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹⁵N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-(13)C, (15)N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during (15)N-(13)C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-(13)C, (15)N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils.

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15

Extracellular electron transfer is one of the physiological hallmarks of Geobacteraceae. Most of the Geobacter species encode for more than 100 c-type cytochromes which are, in general, poorly conserved between individual species. An exception to this is the PpcA family of periplasmic triheme c-type cytochromes, which are the most abundant proteins in these bacteria. The functional characterization of PpcA showed that it has the necessary properties to couple electron/proton transfer, a fundamental step for ATP synthesis. The detailed thermodynamic characterization of a PpcA mutant, in which the strictly conserved residue phenylalanine 15 was replaced by leucine, showed that the global redox network of cooperativities among heme groups is altered, preventing the mutant from performing a concerted electron/proton transfer. In this work, we determined the solution structure of PpcA F15L mutant in the fully reduced state using NMR spectroscopy by producing 15N-labeled protein. In addition, pH-dependent conformational changes were mapped onto the structure. The mutant structure obtained is well defined, with an average pairwise root-mean-square deviation of 0.36Å for the backbone atoms and 1.14Å for all heavy atoms. Comparison between the mutant and wild-type structures elucidated the contribution of phenylalanine 15 in the modulation of the functional properties of PpcA.

Cell-free synthesis system suitable for disulfide-containing proteins

Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional 15N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

Metabolism of soluble rapeseed meal (Brassica rapa L.) protein during incubations with buffered bovine rumen contents in vitro

Accurate quantitative information on the fate of dietary protein in the rumen is central to modern metabolizable protein systems developed to improve the efficiency of nitrogen utilization in ruminants. An in vitro method was developed to estimate the rate of soluble rapeseed meal (Brassica rapa L.) protein (SRMP) degradation. Unlabeled and 15N-labeled solvent-extracted rapeseed meal were incubated alone or as an equal mixture (125mg of N/L) with buffered rumen contents and a mixture of carbohydrates formulated to provide a constant source of fermentable energy during the course of all incubations. Incubations were made over 0.33, 0.67, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, and 10.0h. Enrichment of 14N and 15N isotopes in total N of ammonia (AN), soluble nonammonia (SNAN), and insoluble (ISN) fractions liberated during incubations with test proteins was determined. A model with 4 pools that accounted for both intracellular and extracellular N transformations was used to estimate the rate of SRMP degradation. Parameter values used in the model were adjusted based on the size of A14N, A15N, SNA14N, SNA15N, IS14N, and IS15N pools, measured at different time points during incubations with buffered rumen fluid. The mean rate of N degradation for SRMP was estimated at 0.126 (SD 0.0499) h−1. No substantive difference in the rate of protein degradation or microbial protein synthesis was observed during incubations of labeled and unlabeled substrates with rumen fluid. In conclusion, combined use of data from incubations of unlabeled and 15N-labeled rapeseed protein with buffered rumen inoculum provided sufficient information to allow for estimation of parameter values in a complex dynamic model of soluble protein degradation. Results indicate the potential of the technique to evaluate the degradability of SNAN of other dietary protein sources and implicate ruminal escape of soluble rapeseed protein as an important source of amino acids in ruminants.

Identification and Quantification of Human DNA Repair Proteins: OGG1 and NEIL1 by Liquid Chromatography/isotope-Dilution Tandem Mass Spectrometry Using 15N-Labeled Whole Proteins as Internal Standards

Identification and Quantification of Human DNA Repair Proteins: OGG1 and NEIL1 by Liquid Chromatography/isotope-Dilution Tandem Mass Spectrometry Using 15N-Labeled Whole Proteins as Internal Standards

Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target's topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in (2)H/(13)C/(15)N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data.