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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
NASA NTRS2006

Fluorescent Approaches to High Throughput Crystallography

We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Pusey, Marc L.↗
DOE OSTI2020

Detecting differential protein abundance by combining peptide level P -values

The majority of methods for detecting differentially abundant proteins between samples in label-free LC-MS bottom-up proteomics experiments rely on statistically testing inferred protein abundances derived from peptide ionization intensities or averaging peptide level statistics. Here in this paper, we statistically test peptide ionization intensities directly and combine the resulting dependent P-values using the Empirical Brown's Method (EBM), avoiding error introduced through the estimation of protein abundances or summarizing test statistics. We show that on a spike-in proteomics dataset, a peptide level approach using EBM outperforms differential abundance detection using a protein level approach and several analysis workflows, including MSstats. Additionally, we demonstrate the effectiveness of this approach by detecting enriched proteins from an activity-based protein profiling dataset.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

Non-NASA Center↗
NASA NTRS1997

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Non-NASA Center↗
NASA NTRS1995

Neurofilament protein defines regional patterns of cortical organization in the macaque monkey visual system: a quantitative immunohistochemical analysis

Visual function in monkeys is subserved at the cortical level by a large number of areas defined by their specific physiological properties and connectivity patterns. For most of these cortical fields, a precise index of their degree of anatomical specialization has not yet been defined, although many regional patterns have been described using Nissl or myelin stains. In the present study, an attempt has been made to elucidate the regional characteristics, and to varying degrees boundaries, of several visual cortical areas in the macaque monkey using an antibody to neurofilament protein (SMI32). This antibody labels a subset of pyramidal neurons with highly specific regional and laminar distribution patterns in the cerebral cortex. Based on the staining patterns and regional quantitative analysis, as many as 28 cortical fields were reliably identified. Each field had a homogeneous distribution of labeled neurons, except area V1, where increases in layer IVB cell and in Meynert cell counts paralleled the increase in the degree of eccentricity in the visual field representation. Within the occipitotemporal pathway, areas V3 and V4 and fields in the inferior temporal cortex were characterized by a distinct population of neurofilament-rich neurons in layers II-IIIa, whereas areas located in the parietal cortex and part of the occipitoparietal pathway had a consistent population of large labeled neurons in layer Va. The mediotemporal areas MT and MST displayed a distinct population of densely labeled neurons in layer VI. Quantitative analysis of the laminar distribution of the labeled neurons demonstrated that the visual cortical areas could be grouped in four hierarchical levels based on the ratio of neuron counts between infragranular and supragranular layers, with the first (areas V1, V2, V3, and V3A) and third (temporal and parietal regions) levels characterized by low ratios and the second (areas MT, MST, and V4) and fourth (frontal regions) levels characterized by high to very high ratios. Such density trends may correspond to differential representation of corticocortically (and corticosubcortically) projecting neurons at several functional steps in the integration of the visual stimuli. In this context, it is possible that neurofilament protein is crucial for the unique capacity of certain subsets of neurons to perform the highly precise mapping functions of the monkey visual system.

Non-NASA Center↗
DOE OSTI2020

DiB-splits: nature-guided design of a novel fluorescent labeling split system

Fluorogen-activating proteins (FAPs) are innovative fluorescent probes combining advantages of genetically-encoded proteins such as green fluorescent protein and externally added fluorogens that allow for highly tunable and on demand fluorescent signaling. Previously, a panel of green- and red-emitting FAPs has been created from bacterial lipocalin Blc (named DiBs). Here we present a rational design as well as functional and structural characterization of the first self-assembling FAP split system, DiB-splits. This new system decreases the size of the FAP label to ~8–12 kDa while preserving DiBs’ unique properties: strong increase in fluorescence intensity of the chromophore upon binding, binding affinities to the chromophore in nanomolar to low micromolar range, and high photostability of the protein-ligand complex. These properties allow for use of DiB-splits for wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also represent an attractive starting point for further design of a protein-protein interaction detection system as well as novel FAP-based sensors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A large expert-curated cryo-EM image dataset for machine learning protein particle picking

Cryo-electron microscopy (cryo-EM) is a powerful technique for determining the structures of biological macromolecular complexes. Picking single-protein particles from cryo-EM micrographs is a crucial step in reconstructing protein structures. However, the widely used template-based particle picking process is labor-intensive and time-consuming. Though machine learning and artificial intelligence (AI) based particle picking can potentially automate the process, its development is hindered by lack of large, high-quality labelled training data. To address this bottleneck, we present CryoPPP, a large, diverse, expert-curated cryo-EM image dataset for protein particle picking and analysis. It consists of labelled cryo-EM micrographs (images) of 34 representative protein datasets selected from the Electron Microscopy Public Image Archive (EMPIAR). The dataset is 2.6 terabytes and includes 9,893 high-resolution micrographs with labelled protein particle coordinates. The labelling process was rigorously validated through 2D particle class validation and 3D density map validation with the gold standard. The dataset is expected to greatly facilitate the development of both AI and classical methods for automated cryo-EM protein particle picking.

42 ENGINEERING↗
NASA NTRS1985

Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

Silver, Geri↗
DOE OSTI2026

Proteome-wide analysis of protein stability in Escherichia coli under acid stress

Knowledge of protein acid sensitivity remains sparse and is largely derived from low-throughput, enzyme-specific assays. We used a scalable framework to map acid stability across the Escherichia coli proteome to assess the acid stability of 1,675 unique proteins, estimating pH 50 values for over 90% of them. The parameter pH50 was defined as the pH value at which only 50% of the initial protein remains in solution following acid treatment. Proteome-wide pH 50 values ranged from 2.28 to 6.33 (median 5.11). Approximately 9% of detected proteins remained stable across all tested pH conditions. Our results align with published data and the assay of citrate synthase (GltA) performed here. Protein acid stability differed significantly by subcellular localization: periplasmic proteins were relatively more abundant in the acid-stable group, cytoplasmic proteins were abundant at pH 50 values 4.5–5.5, and inner membrane proteins at higher pH 50 between 5.5 and 6.0. Outer membrane proteins were too few to draw strong conclusions regarding enrichment within specific pH 50 groups. Notably, the periplasmic binding protein of the molybdate ABC transporter (ModA), was enriched after incubation at low pH. Estimated pH 50 values showed no correlation with protein isoelectric point and molecular weight. Together, this work provides the first proteome-wide map of protein acid stability and establishes a general framework for studying different chemical stressors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

Branched-chain amino acid supplementation during bed rest: effect on recovery

Bed rest is associated with a loss of protein from the weight-bearing muscle. The objectives of this study are to determine whether increasing dietary branched-chain amino acids (BCAAs) during bed rest improves the anabolic response after bed rest. The study consisted of a 1-day ambulatory period, 14 days of bed rest, and a 4-day recovery period. During bed rest, dietary intake was supplemented with either 30 mmol/day each of glycine, serine, and alanine (group 1) or with 30 mmol/day each of the three BCAAs (group 2). Whole body protein synthesis was determined with U-(15)N-labeled amino acids, muscle, and selected plasma protein synthesis with l-[(2)H(5)]phenylalanine. Total glucose production and gluconeogenesis from alanine were determined with l-[U-(13)C(3)]alanine and [6,6-(2)H(2)]glucose. During bed rest, nitrogen (N) retention was greater with BCAA feeding (56 +/- 6 vs. 26 +/- 12 mg N. kg(-1). day(-1), P < 0.05). There was no effect of BCAA supplementation on either whole body, muscle, or plasma protein synthesis or the rate of 3-MeH excretion. Muscle tissue free amino acid concentrations were increased during bed rest with BCAA (0.214 +/- 0.066 vs. 0.088 +/- 0.12 nmol/mg protein, P < 0.05). Total glucose production and gluconeogenesis from alanine were unchanged with bed rest but were significantly reduced (P < 0.05) with the BCAA group in the recovery phase. In conclusion, the improved N retention during bed rest is due, at least in part, to accretion of amino acids in the tissue free amino acid pools. The amount accreted is not enough to impact protein kinetics in the recovery phase but does improve N retention by providing additional essential amino acids in the early recovery phase.

Non-NASA Center↗
NASA NTRS1990

Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

Daunton, N.↗
NASA NTRS2007

Recombinant Collagenlike Proteins

A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

Fertala, Andzej↗
NASA NTRS1994

The evolution of energy-transducing systems. Studies with archaebacteria

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of C-14 NEM is incorporated into subunit one. Cyanogen bromide cleavage of labeled subunit one indicated that NEM bound to a peptide of a Mr of about 8,900. Thus, Cys 262 (H. salinarium numbering) may be the NEM binding site. Cyanogen bromide fragments have been submitted for sequencing. To prove the presence of three Cys residues in subunit one, alkaline cleavage following treatment with NTCB was carried out. Thiol reagents such as p-chloro mercuri phenyl sulfonate also inhibited the ATPase. However, this inhibition was not nucleotide-protectable, suggesting a different location and role for the PCMS-sensitive Cys. The proteolipid which was extracted with chloroform/methanol from the membranes of H. saccharovorum cross-reacted with an antiserum against subunit c (the DCCD-binding protein) of Escherichia coli. Following labeling of membranes from H. saccharovorum with C-14 DCCD under conditions, which inhibited ATP synthesis, the isotope was incorporated into one protein of Mr of about 6,500. Thus, the proteolipid of H. saccharovorum and the DCCD-labeled peptide may be identical. If so, these results suggest that the proteolipid is a component of the membrane sector of an archaeal F-type ATP synthase.

Stan-Lotter, Helga↗
DOE OSTI2024

Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dynamic entity formed by protein and its hydration water

The interaction between protein and water plays a pivotal role in shaping the structure, dynamics, and function of biomacromolecules. A comprehensive understanding of this intricate interplay necessitates a systematic evaluation of interaction strength and its consequential impact on the dynamics of proteins and water across diverse protein systems. Despite numerous works on understanding the dynamics of water and proteins and the coupling between them, there are still unanswered questions. Here, we combine neutron scattering and isotope labeling to probe the dynamics of proteins and their hydration water in a variety of protein systems. We consider proteins of different structures and varying thermostability as well as proteins within living cells with distinct growth temperatures. Simultaneous characterization of protein and hydration water dynamics across diverse systems was achieved. Moreover, we performed water sorption isothermal measurements on three representative proteins to correlate the observed dynamics with the strength of the interaction energies governing each system. The experimental results underscore that proteins manifesting stronger attractive interactions with water display diffusionlike dynamics with higher flexibility upon hydration, concomitant with a reduced mobility in hydration water. Significantly, our findings suggest that, in fact, it is the interaction between protein and its hydration water that facilitates the transfer of mobility from water to protein, with stronger interactions correlating to greater protein flexibility and slower hydration water diffusion. Published by the American Physical Society 2024

Ye, Yongfeng (ORCID:0009000792374198)↗
NASA NTRS1999

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Rosenthal, Nadia A.↗
DOE OSTI2022

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.

31P-NMR↗